Modified pyrazine derivatives and uses thereof

ABSTRACT

Provided herein are compounds, preparations and formulations comprising pyrazine derivatives having multiple poly(ethylene glycol) containing substituents. Many of the compounds disclosed herein are excretable by the renal system of a subject or patient and are useful for visualizing the renal system of a subject or patient. Upon excitation by electromagnetic radiation, a number of the compounds disclosed herein exhibit luminescence and are externally detectable when present in the body fluid of a patient or subject. Also provided herein are methods for visualizing the renal system of a subject or patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/140,082 filed Jun. 16, 2011, which is a national stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US09/68453,filed Dec. 17, 2009, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/138,149 filed on Dec. 17, 2008, and U.S.Provisional Patent Application Ser. No. 61/139,911 filed on Dec. 22,2008, each of which are hereby incorporated by reference in theirentirety.

BACKGROUND

The present invention relates to pyrazine derivatives and methods ofusing the same in medical procedures.

As a preliminary note, various publications are referenced throughoutthis disclosure by Arabic numerals in brackets. A citation correspondingto each reference number is listed following the detailed description.

Acute renal failure (ARF) is a common ailment in patients admitted togeneral medical-surgical hospitals. Approximately half of the patientswho develop ARF die, and survivors face marked increases in morbidityand prolonged hospitalization [1]. Early diagnosis is generally believedto be important, because renal failure is often asymptomatic andtypically requires careful tracking of renal function markers in theblood. Dynamic monitoring of patient renal function is desirable inorder to reduce the risk of acute renal failure brought about by variousclinical, physiological and pathological conditions [2-6]. Such dynamicmonitoring might be particularly desirable in the case of critically illor injured patients, because a large percentage of these patients tendto face risk of multiple organ failure (MOF) potentially resulting indeath [7, 8]. MOF is a sequential failure of the lungs, liver andkidneys and is incited by one or more of acute lung injury (ALI), adultrespiratory distress syndrome (ARDS), hypermetabolism, hypotension,persistent inflammatory focus and sepsis syndrome. Common histologicalfeatures of hypotension and shock leading to MOF generally includetissue necrosis, vascular congestion, interstitial and cellular edema,hemorrhage and microthrombi. These changes generally affect the lungs,liver, kidneys, intestine, adrenal glands, brain, and pancreas indescending order of frequency [9]. The transition from early stages oftrauma to clinical MOF generally corresponds with a particular degree ofliver and renal failure as well as a change in mortality risk from about30% up to about 50% [10].

Traditionally, renal function of a patient has been determined usingcrude measurements of the patient's urine output and plasma creatininelevels [11-13]. These values may be misleading because such values areaffected by age, state of hydration, renal perfusion, muscle mass,dietary intake, and many other clinical and anthropometric variables. Inaddition, a single value obtained several hours after sampling may bedifficult to correlate with other physiologic events such as bloodpressure, cardiac output, state of hydration, and other specificclinical events (e.g., hemorrhage, bacteremia, ventilator settings andothers).

With regard to conventional renal monitoring procedures, anapproximation of a patient's glomerular filtration rate (GFR) may bemade via a 24 hour urine collection procedure that (as the namesuggests) typically requires about 24 hours for urine collection,several more hours for analysis, and a meticulous bedside collectiontechnique. Unfortunately, the undesirably late timing and significantduration of this conventional procedure may reduce the likelihood ofeffectively treating the patient and/or saving the kidney(s). As afurther drawback to this type of procedure, repeat data tends to beequally as cumbersome to obtain as the originally acquired data.

Occasionally, changes in serum creatinine of a patient are adjustedbased on measurement values such as the patient's urinary electrolytesand osmolality as well as derived calculations such as “renal failureindex” and/or “fractional excretion of sodium.” Such adjustments ofserum creatinine undesirably tend to require contemporaneous collectionof additional samples of serum and urine and, after some delay, furthercalculations. Frequently, dosing of medication is adjusted for renalfunction and thus may be equally as inaccurate, equally delayed, and asdifficult to reassess as the measurement values and calculations uponwhich the dosing is based. Finally, clinical decisions in the criticallyill population are often equally as important in their timing as theyare in their accuracy.

It is known that hydrophilic, anionic substances are generally capableof being excreted by the kidneys [14]. Renal clearance typically occursvia two pathways: glomerular filtration and tubular secretion. Tubularsecretion may be characterized as an active transport process, andhence, the substances clearing via this pathway typically exhibitspecific properties with respect to size, charge and lipophilicity.

Most of the substances that pass through the kidneys are filteredthrough the glomerulus (a small intertwined group of capillaries in themalpighian body of the kidney). Examples of exogenous substances capableof clearing the kidney via glomerular filtration (hereinafter referredto as “GFR agents”) are shown in FIG. 1 and include creatinine,o-iodohippuran, and ^(99m)Tc-DTPA [15-17]. Examples of exogenoussubstances that are capable of undergoing renal clearance via tubularsecretion include ^(99m)Tc-MAG3 and other substances known in the art[15, 18, 19]. ^(99m)Tc-MAG3 is also widely used to assess renal functionthough gamma scintigraphy as well as through renal blood flowmeasurement. As one drawback to the substances illustrated in FIG. 1,o-iodohippuran, ^(99m)Tc-DTPA and ^(99m)Tc-MAG3 include radioisotopes toenable the same to be detected. Even if non-radioactive analogs (e.g.,such as an analog of o-iodohippuran) or other non-radioactive substanceswere to be used for renal function monitoring, such monitoring wouldtypically require the use of undesirable ultraviolet radiation forexcitation of those substances.

SUMMARY

The present invention generally relates to compounds for biomedicalapplications, including imaging and diagnosing medical conditions.Compounds provided absorb and emit spectral energy in the visible, nearinfrared, and/or any other wavelength range useful for optical detectionin medical procedures. The invention includes compounds and relatedtherapeutic methods, comprising pyrazine compounds having substituentgroups which allow tailoring of the spectral properties and/or providedesired characteristics in a biological system.

In embodiments, provided are compounds and methods useful for measuringproperties of the renal system, including renal excretion rate and renalfunction. In an aspect, the invention relates to compounds useful forrapid, and in an embodiment, real-time, assessment of renal function.Compounds of the invention are generally characterized as pyrazinederivatives. Pyrazine derivatives of the invention are desirable forrenal applications because they are cleared from the body via thekidneys, demonstrate strong absorption and luminescence in usefulregions of the electromagnetic spectrum and exhibit significant Stokesshifts. In an embodiment, for example, an absorption maxima of acompound of the invention is shifted a selected number of nanometers(e.g., 10-50 nm) toward the red region of the electromagnetic spectrumby selection of the composition of substituents of the central pyrazinegroup. These properties provide flexibility in both tuning the moleculeto the desired absorption and emission wavelengths and providingsubstituents to improve renal clearance properties. In an aspect,pyrazine compounds of the present invention have renal clearanceproperties that are comparable with or better than that of creatinine oriothalamate or other conventional renal clearance assessment agents.

In an aspect, the present invention is directed to pyrazine derivativesof Formula (FX1):

wherein:

R¹ and R³ are each independently —H, —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵,—(CH₂CH₂O)_(b)R⁵, —CH(COOH)CH₂OH or —(CH₂)_(a)Y¹;

each Y¹ is independently —OR⁶, —(CHOH)_(c)R⁷, —NR⁸R⁹, —CONR⁸R⁹,—NHCO(CHOH)_(c)R⁷ or —NHCO(CH₂)_(a)(CH₂CH₂O)_(b)R⁵;

each of R², R⁴, R⁵, R⁶ and R⁷ are independently —H or C₁-C₆ alkyl;

R⁸ and R⁹ are independently —H, C₁-C₃ alkyl, —(CH₂)_(a)(CHOH)_(c)R⁷, or—(CH₂)_(a)(CH₂CH₂O)_(b)R⁵;

each a and c is independently an integer selected from the range of 0 to6;

each b is independently an integer selected from the range of 1 to 120;

each p and q is independently an integer selected from the range of 0 to120;

each of m and n is independently an integer selected from the range of 3to 6.

In one aspect, described herein are compounds of Formula (FX1) and theirpharmaceutically acceptable salts and esters. In some embodiments, eachof R¹ and R³ is —CH(COOH)CH₂OH. In other embodiments, each of R¹ and R³is —(CH₂CH₂O)_(b)R⁵. In still other embodiments, each of R¹ and R³ is—H. In other embodiments, each of R¹ and R³ is —(CH₂)_(a)Y¹. In evenother embodiments, each of R¹ and R³ is —CH₃. In still otherembodiments, each of R¹ and R³ is —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵.

Still referring to compounds of Formula (FX1), in one embodiment R², R⁴,R⁵, R⁶ and R⁷ are each independently —H or C₁-C₃ alkyl. In someembodiments, each occurrence of R⁵ and R⁷ is independently C₁-C₆ alkyl(e.g., each occurrence of R⁵ is C₁ alkyl). In some embodiments, each ofR² and R⁴ is —H. In other embodiments, each of R² and R⁴ isindependently C₁-C₃ alkyl (e.g., each of R² and R⁴ is C₁ alkyl).

The integer m independently varies from 3 to 6, inclusive. For instance,in some embodiments, m may be 3 or 4 (e.g., m may be 3 in someembodiments). Likewise, the integer n independently varies from 3 to 6,inclusive. For instance, in some embodiments, n may be 3 or 4 (e.g., nmay be 3 in some embodiments). One of the benefits of m and nindependently varying from 3 to 6 is that the pyrazine derivative can be“tuned” to absorb or luminesce at a desired wavelength or range ofwavelengths. In this regard, a pyrazine derivative having both m and nequal to 3 may absorb and/or luminesce at respective light wavelengthsthat are greater than (e.g., about 10-15 nm greater than) that of agenerally similar pyrazine derivative where both m and n are equal to 2.A similar phenomenon could be observed moving from 3 to 4, from 4 to 5,and/or from 5 to 6. Accordingly, pyrazine derivatives described hereinmay be designed to absorb and/or luminesce at light wavelengths that maypenetrate tissues better than that of lower light wavelengths.

Still referring to compounds of Formula (FX1), each occurrence of p andq independently varies from 0 to 120, inclusive. In some embodiments,each occurrence of p and q independently varies from 1 to 120,inclusive. For instance, in some embodiments, each of p and qindependently varies from 2 to 50, inclusive. In other embodiments, eachof p and q independently varies from 2 to 24, inclusive. In otherembodiments, each of p and q independently varies from 1 to 99,inclusive. In other embodiments, each of p and q independently variesfrom 2 to 40, inclusive. In other embodiments, each of p and qindependently varies from 3 to 23, inclusive. In embodiments, the numberof PEG groups in a particular chain varies from 1 to 120. In otherembodiments, the number of PEG groups in a particular chain varies from2 to 50. In other embodiments, the number of PEG groups in a particularchain varies from 2 to 24.

With regard to the various possibilities for R¹ and R³ in Formula (FX1),each occurrence of a and c independently varies from 0 to 6, inclusive.For example, each occurrence of a and c may be 3 or 4 in someembodiments. Further, each occurrence of b independently varies from 1to 120, inclusive. For instance, in some embodiments, each occurrence ofb may independently vary from 2 to 50, inclusive. In embodiments, each bis independently an integer from 1 to 100. In embodiments, each b isindependently an integer from 12 to 24. In embodiments, each b isindependently an integer from 10 to 40. In embodiments, each b isindependently an integer from 2 to 24.

In an embodiment, the invention provides compounds having formulae (FX2)to (FX3):

In an embodiment, the invention provides compounds having formulae (FX4)to (FX5):

In an embodiment, the invention provides compounds having formulae (FX6)to (FX17):

In an embodiment, the invention provides compounds having formula(FX18):

where d and h are independently integers selected from the range of 1 to50. In an embodiment, d and h are independently integers selected fromthe range of 2 to 50. In an aspect of the compound of formula (FX18),each a is 2. In an aspect of the compound of formula (FX18), R⁸ and R⁹are each —(CH₂)_(a)(CHOH)_(c)R⁷.

In an embodiment, the invention provides compounds having formula(FX19):

where d and h are independently integers selected from the range of 1 to50. In an embodiment, d and h are independently integers selected fromthe range of 2 to 50. In an aspect of the compound of formula (FX19), Y¹is —NR⁸R⁹ wherein R⁸ and R⁹ are each —(CH₂)_(a)(CHOH)_(c)R⁷ wherein eacha is independently 1 or 2 and c is 2, 3, 4, 5 or 6.

In an embodiment, the invention provides compounds having formula(FX20):

where d and h are independently integers selected from the range of 1 to50. In an embodiment, d and h are independently integers selected fromthe range of 2 to 50. In a specific aspect of the compound of formula(FX20), each a is independently 0, 2 or 3, and each R⁵ is independentlyC₁-C₃ alkyl. In an embodiment, the invention provides compounds havingformula (FX20-1) having the formula:

where each z is independently an integer selected from the range of 0 to6.

In an embodiment, the invention provides compounds having formula(FX21):

wherein d and h are independently integers selected from the range of 1to 50 and each a is independently an integer selected from the range of0 to 6. In an embodiment, d and h are independently integers selectedfrom the range of 2 to 50.

In an embodiment, the invention provides compounds having formula (FX22)

where d and h are independently integers from 1 to 50. In an embodiment,d and h are independently integers selected from the range of 2 to 50.

In an embodiment, the invention provides compounds having formula(FX23):

where d and h are independently integers selected from the range of 1 to50. In an embodiment, d and h are independently integers selected fromthe range of 2 to 50.

In an embodiment, the invention provides compounds having formula (FX24)

where d and h are independently integers from 1 to 50 and each b isindependently an integer selected from the range of 1 to 50. In anembodiment, d and h are independently integers selected from the rangeof 2 to 50.

In an embodiment, the invention provides compounds having formula(FX25):

where d and h are independently integers selected from the range of 1 to50. In an embodiment, d and h are independently integers selected fromthe range of 2 to 50.

In an aspect of the invention, p and q are the same integer. In anaspect of the invention, each b appearing in a formula is the sameinteger. In an aspect of the invention, each b appearing in a formula isa different integer. In an aspect of the invention, each b appearing ina formula is the same or different integer. In an aspect of theinvention, each alphabetical variable (i.e., a, b, p, q, d, h, c, m, n,etc.) is the same or different integers. In an aspect of the invention,each c appearing in a formula is the same integer. In an aspect of theinvention, each a appearing in a —(CH₂)_(a)Y¹ group is the same integer.In an aspect of the invention, R² and R⁴ are the same. In an aspect ofthe invention, the groups that are para to each other on the pyrazinering are the same. In an aspect of the invention, R¹ and R³ are thesame. In an aspect of the invention, m and n are the same. In an aspectof the invention, compounds of the invention are aminopyrazine compoundshaving one or more poly(ethylene glycol) (PEG) groups. In an aspect ofthe invention, PEG groups add hydrophilic character to the pyrazinederivatives. As used herein, PEG groups are generally depicted by theformula —(CH₂CH₂O)_(b)— and are also known as repeating ethylene oxidegroups.

In an embodiment, compounds of the invention comprise one or morebranched or straight chain alkyl groups containing two or more hydroxylgroups. These groups can be referred to as “polyhydroxylated alkyl” or“polyhydroxyalkyl” groups in an embodiment. In an embodiment, thepolyhydroxylated alkyl group has from three to six carbon atoms. In anembodiment, the polyhydroxylated alkyl group includes two or more—(CHOH) groups along with one or more methylene groups, depicted by theformula: —(CHR¹⁰)_(r)R¹¹ where R¹⁰ and R¹¹ are each independently H,C₁-C₃ alkyl or —OH, and r is an integer from 1 to 10. In an embodiment,the hydroxyl groups may be adjacent to each other or separated by one ormore methylene groups. In an embodiment, the polyhydroxylated alkylgroup refers to a substituent having from 2 to 12 carbon atoms and from2 to 5 hydroxyl groups, such as the 2,3-dihydroxypropyl,2,3,4-trihydroxybutyl or 2,3,4,5-tetrahydroxypentyl residue.

In an aspect of the invention, there is a three to six carbon alkylenegroup attached to the amino groups directly attached to the pyrazinecore.

In an embodiment, compounds absorb electromagnetic radiation havingwavelengths in the UVA, visible and near IR portion of the spectrum. Inan embodiment, compounds of the invention absorb electromagneticradiation between 350 to 1300 nm. In an embodiment, compounds of theinvention absorb electromagnetic radiation having wavelengths between400 to 900 nm. In an embodiment, compounds of the invention absorbelectromagnetic radiation having wavelengths between 300 to 500 nm. Inan embodiment, the compounds of the invention exhibit luminescencebetween 520 to 650 nm. In an aspect of the invention, it is desirablefor the compounds to absorb and emit at longer wavelengths to enhanceoptical detection methods due to enhanced tissue penetration fromminimized absorption from hemoglobin, water, lipids and other substancespresent in a biological system.

In an aspect of the invention, tetra-substituted pyrazine derivatives ofthe invention having PEG groups are typically hydrophilic. Increasingthe number of PEG groups on the pyrazine derivatives is believed toincrease the terminal half-life in circulation of the body. It isbelieved that lower molecular weight PEG chains (<6000 Da) are filteredby the glomerulus and not absorbed by renal tubules. Thesecharacteristics are useful in tailoring the compound to have the desiredcharacteristics.

In an aspect of the invention, compounds of the invention have percentplasma binding of 20% or less. In an aspect of the invention, compoundsof the invention have percent plasma binding of 15% or less. In anaspect of the invention, compounds of the invention have percent plasmabinding of 10% or less. In an aspect of the invention, compounds of theinvention have percent plasma binding of 1-10%. In an aspect of theinvention, compounds of the invention have percent plasma binding of1-15%. As used herein, “percent plasma binding” is the amount ofcompound that binds to plasma upon administration to a subject, such asa human subject. In an embodiment of the invention, compounds of theinvention are highly cleared in the urine in a patient within a periodof time of hours. In a particular embodiment, compounds of the inventionare at least 80% cleared in the urine within 10 hours afteradministration.

In an aspect of the present invention, a compound of the invention isused in an optical imaging, biomedical imaging, diagnostic,visualization, monitoring, surgical, biomedical or therapeutic procedureon a patient. In an aspect of the present invention, the invention isdirected to performing an optical imaging, biomedical imaging,diagnostic, visualization, monitoring, surgical, biomedical ortherapeutic procedure on a patient. In an aspect of the presentinvention, the invention is directed to a method of performing abiomedical imaging procedure or diagnostic procedure on a patient. In anaspect, the method comprises administering a diagnostically effectiveamount of the compound to a subject, wherein the compound isdifferentially separated from a bodily fluid of the subject by an organ,tissue or system in the subject; and detecting the administeredcompound. In an aspect, the compound can be detected chemically, where asample of a bodily fluid is analyzed after administration of thecompound and the concentration of the compound is measured. In anembodiment of this aspect of the invention, the compound is administeredinto a bodily fluid.

In an aspect of the present invention, the biomedical imaging procedureor diagnostic procedure comprises detecting electromagnetic radiationemitting or luminescing from the compound in the subject. In an aspectof the biomedical imaging procedure or diagnostic procedure of thepresent invention, exposing the compound administered to the subject toelectromagnetic radiation changes an optical property of the compound.In an aspect of the invention, the change in optical property of thecompound administered from exposure to electromagnetic radiation ismeasured or monitored.

In an aspect of the invention, the electromagnetic radiation isnonionizing. In an aspect of the invention, exposing the administeredcompound to electromagnetic radiation generates luminescence from thecompound, for example fluorescence. In an aspect of the invention, theprocedure comprises detecting luminescence from the administeredcompound. In an aspect of the invention, the method comprises generatingan image based, at least in part on the luminescence from the compound.In an aspect of the invention, the luminescence is collected proximateto the subject's ear, hand, head, forehead, or finger. In an aspect ofthe invention, the luminescence is detected visually. In an aspect ofthe invention, the luminescence is detected using a camera, chargedcoupled device, or diode array. In an aspect of the invention, thebiomedical imaging or diagnosing procedure comprises: administering aneffective amount of a renally excretable compound of the invention to asubject; exposing a tissue of the subject's renal system having theadministered compound to electromagnetic radiation, thereby generatingemitted electromagnetic radiation from the compound; detecting theemitted electromagnetic radiation from the compound, thereby visualizingor imaging at least a portion of the renal system of the subject. In anaspect of the invention, the procedure comprises determining if theadministered compound is substantially retained in tissue of thesubject's renal system. In an aspect of the invention, provided is acompound described herein for use in a biomedical procedure forassessing physiological function of an organ, tissue or system. In anaspect of the invention, a compound of the invention is used in vivo inassessing renal function of a subject. In an aspect of the invention, acompound of the invention is used in vivo in detecting at least aportion of the urinary system of a subject in a surgical procedure. Inan aspect of the invention, a portion of the urinary system comprises aureter, bladder or urethra of the subject. In an aspect of theinvention, a compound of the invention is: administered into thebloodstream of a subject; exposed to electromagnetic radiation while inthe bloodstream of the subject; and detected within the bloodstream ofthe subject. In an aspect of the invention, provided is a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable excipient. In an aspect of the invention,provided is a compound of the invention and one or more additionaltherapeutic agents or diagnostic agents.

The present invention further includes compositions for biomedicalapplications, including monitoring renal function comprising purifiedstereoisomers (e.g., enantiomers and diastereomers), salts (includingquarternary salts), and/or ionic forms (e.g., protonated anddeprotonated forms) of the compounds of any of formula (FX1)-(FX25), andmixtures thereof, and related methods of using compounds of formulae(FX1) to (FX25), for example in a biomedical procedure. As will beunderstood by those having general skill in the art, acidic functionalgroups and basic functional groups of the compounds of any of formula(FX1)-(FX25) may be in protonated or deprotonated states depending onthe molecular environment (e.g., pH, ionic strength, composition, etc.),for example during synthesis, formulation and/or administration

The present invention provides methods of making and using compounds ofthe invention, including specifically those shown herein, in particularcompounds of formulas (FX1) to (FX25). Methods of this aspect of thepresent invention include in vivo, in vitro and ex vivo methods forbiomedical and bioanalytical applications. Methods of the presentinvention include photodiagnostic and phototherapeutic methods, such asoptical imaging, anatomical visualization, endoscopic visualization, andimage guided surgery. For some compounds for use in vivo, in vitro or exvivo for imaging or visualizing, the tissue, organs and/or cells is akidney, ureter, kidney cell or other portion of the renal system.

In another aspect, described herein are “kits” that include one or morecompounds (or pharmaceutically acceptable salt thereof) having a formula(FX1)-(FX25) and a pharmaceutically acceptable carrier in a singlepackage. In other words, the compound (or pharmaceutically acceptablesalt thereof) and the pharmaceutically acceptable carrier are packagedtogether. A kit of this aspect optionally includes instructions (e.g.,in the form of a paper product insert) for preparing and/or utilizing acomposition that includes the compound (or pharmaceutically acceptablesalt thereof) and the pharmaceutically acceptable carrier. In someembodiments, the compound (or pharmaceutically acceptable salt thereof)may have already been combined with (e.g., dissolved or suspendedwithin) the pharmaceutically acceptable carrier prior to placing thesame within the packaging. In other embodiments, the compound (orpharmaceutically acceptable salt thereof) may be disposed within a firstcontainer, and the pharmaceutically acceptable carrier may be disposedwithin a second container that is separate and distinct from the firstcontainer. In such embodiments, the first and second containers can befound within the same packaging.

Incorporation of a combination of the substituents on the pyrazine ringis particularly beneficial for providing compounds and optical agentshaving large extinction coefficients in the visible and near infraredregions of the electromagnetic spectrum (e.g., 350 nm-1300 nm,optionally 400 nm to 900 nm), emission in the visible and near infraredregions (e.g., 350 nm-1300 nm, optionally 500-900 nm), a largefluorescence quantum yield (e.g., >0.1) and a Stokes shift useful foroptical detection and imaging (e.g., Stokes shift>10 nm). For example,depending on the number of methylene groups present in the substituentslinked to the pyrazine ring, the Stokes shift can be increased by 10 nmor more.

In an embodiment, provided herein are methods for a biomedicalprocedure, such as an imaging procedure, wherein the method comprises:(i) administering (e.g., via intravenous or intraarterial injection,oral administration, topical administration, subcutaneousadministration, etc.) to a subject a diagnostically effective amount ofthe compound having any one of formula (FX1)-(FX25) and (ii) exposingthe administered compound to electromagnetic radiation. In anembodiment, the administering step is carried out under conditionssufficient for contacting the compound with a bodily fluid of thesubject, wherein the compound is differentially separated from thebodily fluid of the subject by an organ, tissue or system in thesubject.

In an embodiment, exposing the administered compound to electromagneticradiation generates a diagnostically effective amount of luminescence,for example an amount of luminescence allowing for optical detection,visualizing and/or imaging of the compound. In an embodiment, a methodof the invention further comprises exposing the administered compound toelectromagnetic radiation having sufficient power, fluence, intensityand/or dose (net number of photons provided to the target tissue) toprovide optical detection, visualization and/or imaging of the compound.In an embodiment, a method of the invention further comprises generatingan image of the luminescence from the compound. In an embodiment, amethod of the invention further comprises visualizing the luminescencefrom the compound.

In an embodiment, the electromagnetic radiation exposed to the compoundof any one of formulas (FX1)-(FX25) does not have wavelengths in theX-ray region of the electromagnetic spectrum. In an embodiment, theelectromagnetic radiation exposed to the compound of any one of formulas(FX1)-(FX25) does not have wavelengths in the ultraviolet region of theelectromagnetic spectrum.

As used herein, pyrazine compounds or derivatives of the presentinvention include a substituted pyrazine group. For example, theinvention provides a pyrazine compound of the Formula A:

where the R variables depict “tails” or substituent groups and arefurther defined and described herein. In a specific embodiment, pyrazinecompounds or derivatives of the present invention include the structureshown in Formula B:

where the R variables are further defined and described herein.

In another aspect, the present invention provides methods forvisualizing body fluids, organs or tissues of a subject. A method ofthis aspect comprises the steps of: administering to a subject aneffective amount of a compound disclosed herein, for example any of thecompounds having formulas (FX1)-(FX25), and detecting the compound in abody fluid, organ or tissue of the subject. In embodiments, the compoundis administered to the subject intravenously, by intraperitoneal orsubcutaneous injection or infusion, by oral administration, bytransdermal absorption through the skin, by inhalation, by parenteraladministration or by any combination of these. In a specific embodiment,the compound is excreted by the subject's renal system, for example fromone body fluid (e.g., blood) into another body fluid (e.g., urine).

In specific embodiments, the step of detecting the compound comprisesexposing the compound in the body fluid, organ or tissue toelectromagnetic radiation, for example having wavelengths selected inthe range of 350 to 1300 nm, 400 to 900 nm or 300 to 500 nm. For certainembodiments, upon excitation by electromagnetic radiation of suitablewavelength, the compounds in the body fluid, organ, tissue or systemexhibit detectable luminescence, for example luminescence havingwavelengths selected in the range of 350 nm to 1300 nm, 500 to 900 nm or520 to 650 nm. In embodiments, the compounds are detected in the bodyfluid, organ or tissue of the subject visually (e.g., by eye) or by acamera, charged coupled device, or diode array. In a specificembodiment, the compounds are detected proximate to the subject's ear,hand, head, forehead, or finger.

Another method of this aspect comprises the steps of: administering to asubject an effective amount of a compound disclosed herein, for exampleany of the compounds having formulas (FX1)-(FX25), and detecting thecompound in a body fluid, organ or tissue of the subject during asurgical procedure. In a specific embodiment, the compound in the bodyfluid, organ or tissue is exposed to electromagnetic radiation, therebygenerating luminescence which is subsequently detected, for examplevisually or by a camera, charged coupled device, or diode array. Theseand other methods are useful, for example, as they can indicate to asurgeon which body fluids, organs or tissues contain the administeredcompound.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesor mechanisms relating to the invention. It is recognized thatregardless of the ultimate correctness of any explanation or hypothesis,an embodiment of the invention can nonetheless be operative and useful.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates structures of some conventional renal agents.

FIG. 2 provides a synthetic scheme for preparation of compounds of thepresent invention.

FIG. 3 provides a synthetic scheme for preparation of compounds of thepresent invention.

FIG. 4 provides a synthetic scheme for preparation of compounds of thepresent invention.

FIG. 5 illustrates a block diagram of an assembly for assessing renalfunction.

FIG. 6 provides a schematic diagram of a monitoring device of thepresent invention.

FIG. 7 illustrates an apparatus for non-invasive in vivo detection offluorescence.

FIG. 8A provides a plot showing non-invasive in vivo fluorescence signalas a function of time after delivery of a renally excretable compound.FIG. 8B provides a plot showing invasive PK (plasma concentration) as afunction of time after delivery of a renally excretable compound.

FIG. 9 provides the plots of FIGS. 8A and 8B plotted versus one anotherto display a linear correlation between the optical and plasmapharmacokinetics.

DETAILED DESCRIPTION

Referring to the drawings, like numerals indicate like elements and thesame number appearing in more than one drawing refers to the sameelement. In general the terms and phrases used herein have theirart-recognized meaning, which can be found by reference to standardtexts, journal references and contexts known to those skilled in theart. The following definitions are provided to clarify their specificuse in the context of the invention.

“Optical agent” generally refers to compounds, compositions,preparations, and/or formulations that absorb, emit, or scatterelectromagnetic radiation of wavelength, generally in the range of350-1300 nanometers, within a biologically relevant environment orcondition. In some embodiments, optical agents of the invention, whenexcited by electromagnetic radiation, undergo emission via luminescencesuch as fluorescence or phosphorescence pathways. These pathways areuseful for diagnostic imaging, visualization, or organ functionmonitoring. Compounds belonging to this class are commonly referred toas ‘optical imaging agents’ or ‘optical contrast agents. In anembodiment, an optical agent is a compound described herein.

Optical agents of the present invention can contain fluorophores. Theterm “fluorophore” generally refers to a component or moiety of amolecule which causes a molecule to be fluorescent. Fluorophores can befunctional groups in a molecule which absorb electromagnetic radiationof first specific wavelengths and re-emit energy at second specificwavelengths. The amount and wavelengths of the emitted electromagneticradiation depend on both the fluorophore and the chemical environment ofthe fluorophore.

Compounds and compositions of the invention provide optical agentsincluding imaging agents and detectable agents; and conjugates,complexes, and derivatives thereof. Some optical agents of the inventionprovide detectable agents that can be administered to a subject andsubsequently detected using a variety of optical techniques, includingoptical imaging, visualization, and one-, two-, three- and point opticaldetection.

Optical agents include, but are not limited to, imaging agents,detectable agents and conjugates, complexes, and derivatives thereof.

When used herein, the terms “diagnosis”, “diagnostic” and other rootword derivatives are as understood in the art and are further intendedto include a general monitoring, characterizing and/or identifying astate of health or disease. The term is meant to encompass the conceptof prognosis. For example, the diagnosis of acute renal failure caninclude an initial determination and/or one or more subsequentassessments regardless of the outcome of a previous finding. The termdoes not necessarily imply a defined level of certainty regarding theprediction of a particular status or outcome.

As defined herein, “administering” means that a compound or formulationthereof of the invention, such as an optical agent, is provided to apatient or subject, for example in a diagnosably effective amount. Theinvention includes methods for a biomedical procedure wherein adiagnostically effective amount of a compound having any one of formulas(FX1)-(FX25) is administered to a patient in need of diagnosis, forexample to a patient suspected of having a disorder in the renal system.Administering may be carried out by a range of techniques known in theart including intravenous, intraperitoneal or subcutaneous injection orinfusion, oral administration, transdermal absorption through the skin,or by inhalation.

Alkyl groups include straight-chain, branched and cyclic alkyl groups.Alkyl groups include those having from 1 to 30 carbon atoms. Alkylgroups include small alkyl groups having 1 to 3 carbon atoms. Alkylgroups include medium length alkyl groups having from 4-10 carbon atoms.Alkyl groups include long alkyl groups having more than 10 carbon atoms,particularly those having 10-30 carbon atoms. Cyclic alkyl groupsinclude those having one or more rings. Cyclic alkyl groups includethose having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbonrings in cyclic alkyl groups can also carry alkyl groups. Cyclic alkylgroups can include bicyclic and tricyclic alkyl groups. Alkyl groups areoptionally substituted. Substituted alkyl groups include among othersthose which are substituted with aryl groups, which in turn can beoptionally substituted. Specific alkyl groups include methyl, ethyl,n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl,cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branchedhexyl, and cyclohexyl groups, all of which are optionally substituted.Substituted alkyl groups include fully halogenated or semihalogenatedalkyl groups, such as alkyl groups having one or more hydrogens replacedwith one or more fluorine atoms, chlorine atoms, bromine atoms and/oriodine atoms. Substituted alkyl groups include fully fluorinated orsemifluorinated alkyl groups, such as alkyl groups having one or morehydrogens replaced with one or more fluorine atoms. An alkoxy group isan alkyl group that has been modified by linkage to oxygen and can berepresented by the formula R—O and may also be referred to as an alkylether group. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, propoxy, butoxy and heptoxy. Alkoxy groups includesubstituted alkoxy groups wherein the alky portion of the groups issubstituted as provided herein in connection with the description ofalkyl groups. As used herein MeO— refers to CH₃O—.

Alkenyl groups include straight-chain, branched and cyclic alkenylgroups. Alkenyl groups include those having 1, 2 or more double bondsand those in which two or more of the double bonds are conjugated doublebonds. Alkenyl groups include those having from 2 to 20 carbon atoms.Alkenyl groups include small alkenyl groups having 2 to 3 carbon atoms.Alkenyl groups include medium length alkenyl groups having from 4-10carbon atoms. Alkenyl groups include long alkenyl groups having morethan 10 carbon atoms, particularly those having 10-20 carbon atoms.Cyclic alkenyl groups include those having one or more rings. Cyclicalkenyl groups include those in which a double bond is in the ring or inan alkenyl group attached to a ring. Cyclic alkenyl groups include thosehaving a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbonrings in cyclic alkenyl groups can also carry alkyl groups. Cyclicalkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenylgroups are optionally substituted. Substituted alkenyl groups includeamong others those which are substituted with alkyl or aryl groups,which groups in turn can be optionally substituted. Specific alkenylgroups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl,but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl,pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branchedhexenyl, cyclohexenyl, all of which are optionally substituted.Substituted alkenyl groups include fully halogenated or semihalogenatedalkenyl groups, such as alkenyl groups having one or more hydrogensreplaced with one or more fluorine atoms, chlorine atoms, bromine atomsand/or iodine atoms. Substituted alkenyl groups include fullyfluorinated or semifluorinated alkenyl groups, such as alkenyl groupshaving one or more hydrogens replaced with one or more fluorine atoms.

Aryl groups include groups having one or more 5-, 6- or 7-memberaromatic or heterocyclic aromatic rings. Aryl groups can contain one ormore fused aromatic rings. Heterocyclic aromatic rings can include oneor more N, O, or S atoms in the ring. Heterocyclic aromatic rings caninclude those with one, two or three N, those with one or two O, andthose with one or two S, or combinations of one or two or three N, O orS. Aryl groups are optionally substituted. Substituted aryl groupsinclude among others those which are substituted with alkyl or alkenylgroups, which groups in turn can be optionally substituted. Specificaryl groups include phenyl groups, biphenyl groups, pyridinyl groups,and naphthyl groups, all of which are optionally substituted.Substituted aryl groups include fully halogenated or semihalogenatedaryl groups, such as aryl groups having one or more hydrogens replacedwith one or more fluorine atoms, chlorine atoms, bromine atoms and/oriodine atoms. Substituted aryl groups include fully fluorinated orsemifluorinated aryl groups, such as aryl groups having one or morehydrogens replaced with one or more fluorine atoms. Aryl groups include,but are not limited to, aromatic group-containing or heterocylicaromatic group-containing groups corresponding to any one of thefollowing benzene, naphthalene, naphthoquinone, diphenylmethane,fluorene, anthracene, anthraquinone, phenanthrene, tetracene,naphthacenedione, pyridine, quinoline, isoquinoline, indoles, isoindole,pyrrole, imidazole, oxazole, thiazole, pyrazole, pyrazine, pyrimidine,purine, benzimidazole, furans, benzofuran, dibenzofuran, carbazole,acridine, acridone, phenanthridine, thiophene, benzothiophene,dibenzothiophene, xanthene, xanthone, flavone, coumarin, azulene oranthracycline. As used herein, a group corresponding to the groupslisted above expressly includes an aromatic or heterocyclic aromaticradical, including monovalent, divalent and polyvalent radicals, of thearomatic and heterocyclic aromatic groups listed above provided in acovalently bonded configuration in the compounds of the invention. Arylgroups optionally have one or more aromatic rings or heterocyclicaromatic rings having one or more electron donating groups, electronwithdrawing groups and/or other ligands provided as substituents.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.Alkylaryl groups are alternatively described as aryl groups substitutedwith one or more alkyl groups wherein the alkyl groups optionally carryadditional substituents and the aryl groups are optionally substituted.Specific alkylaryl groups are alkyl-substituted phenyl groups such asmethylphenyl. Substituted arylalkyl groups include fully halogenated orsemihalogenated arylalkyl groups, such as arylalkyl groups having one ormore alkyl and/or aryl having one or more hydrogens replaced with one ormore fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.

Optional substitution of any alkyl, alkenyl and aryl groups includessubstitution with one or more of the following substituents: halogens,—CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR,—SO₂R, —SO₂N(R)₂ or —SOR groups. Optional substitution of alkyl groupsincludes substitution with one or more alkenyl groups, aryl groups orboth, wherein the alkenyl groups or aryl groups are optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include amongothers:

-   -   —COOR where R is a hydrogen or an alkyl group or an aryl group        and more specifically where R is methyl, ethyl, propyl, butyl,        or phenyl groups all of which are optionally substituted;    -   —COR where R is a hydrogen, or an alkyl group or an aryl groups        and more specifically where R is methyl, ethyl, propyl, butyl,        or phenyl groups all of which groups are optionally substituted;    -   —CON(R)₂ where each R, independently of each other R, is a        hydrogen or an alkyl group or an aryl group and more        specifically where R is methyl, ethyl, propyl, butyl, or phenyl        groups all of which groups are optionally substituted; R and R        can form a ring which may contain one or more double bonds;    -   —OCON(R)₂ where each R, independently of each other R, is a        hydrogen or an alkyl group or an aryl group and more        specifically where R is methyl, ethyl, propyl, butyl, or phenyl        groups all of which groups are optionally substituted; R and R        can form a ring which may contain one or more double bonds;    -   —N(R)₂ where each R, independently of each other R, is a        hydrogen, or an alkyl group, acyl group or an aryl group and        more specifically where R is methyl, ethyl, propyl, butyl, or        phenyl or acetyl groups all of which are optionally substituted;        or R and R can form a ring which may contain one or more double        bonds;    -   —SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups        and more specifically where R is methyl, ethyl, propyl, butyl,        phenyl groups all of which are optionally substituted; for —SR,        R can be hydrogen;    -   —OCOOR where R is an alkyl group or an aryl groups;    -   —SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl        group and R and R can form a ring;    -   —OR where R is H, alkyl, aryl, or acyl; for example, R can be an        acyl yielding —OCOR* where R* is a hydrogen or an alkyl group or        an aryl group and more specifically where R* is methyl, ethyl,        propyl, butyl, or phenyl groups all of which groups are        optionally substituted.

As used herein, the term “alkylene” refers to a divalent radical derivedfrom an alkyl group as defined herein. Alkylene groups in someembodiments function as attaching and/or spacer groups in the presentcompositions. Compounds of the invention include substituted andunsubstituted C₁-C₂₀ alkylene, C₁-C₁₀ alkylene and C₁-C₅ alkylenegroups.

As used herein, the term “cycloalkylene” refers to a divalent radicalderived from a cycloalkyl group as defined herein. Cycloalkylene groupsin some embodiments function as attaching and/or spacer groups in thepresent compositions. Compounds of the invention include substituted andunsubstituted C₁-C₂₀ cycloalkylene, C₁-C₁₀ cycloalkylene and C₁-C₅cycloalkylene groups.

As used herein, the term “alkenylene” refers to a divalent radicalderived from an alkenyl group as defined herein. Alkenylene groups insome embodiments function as attaching and/or spacer groups in thepresent compositions. Compounds of the invention include substituted andunsubstituted C₁-C₂₀ alkenylene, alkenylene and C₁-C₅ alkenylene groups.

As used herein, the term “cycloalkenylene” refers to a divalent radicalderived from a cycloalkenyl group as defined herein. Cycloalkenylenegroups in some embodiments function as attaching and/or spacer groups inthe present compositions. Compounds of the invention include substitutedand unsubstituted C₁-C₂₀ cycloalkenylene, C₁-C₁₀ cycloalkenylene andC₁-C₅ cycloalkenylene groups.

As used herein, the term “alkynylene” refers to a divalent radicalderived from an alkynyl group as defined herein. Alkynylene groups insome embodiments function as attaching and/or spacer groups in thepresent compositions. Compounds of the invention include substituted andunsubstituted C₁-C₂₀ alkynylene, C₁-C₁₀ alkynylene and C₁-C₅ alkynylenegroups.

As used herein, the term “halo” refers to a halogen group such as afluoro (—F), chloro (—Cl), bromo (—Br), iodo (—I) or astato (—At).

As used herein, the term “azo” refers to a group having at least one—N═N— moiety. Azo groups include cyclic and acyclic groups having an—N═N— moiety, for example: (i) aryl-azo groups having an —N═N— moietydirectly or indirectly linked to one or more carbocyclic or heterocyclicaromatic rings of a C₅-C₂₀ aryl, (ii) alkyl-azo groups having an —N═N—moiety directly or indirectly linked to a C₁-C₂₀ alkyl group and (iii)alkylaryl-azo groups having an —N═N— moiety directly or indirectlylinked to a C₁-C₂₀ alkyl group and one or more carbocyclic orheterocyclic aromatic rings of a C₅-C₂₀ aryl. In an embodiment, forexample, an azo group of a compound of the invention includes a cyclicgroup having an intra-ring —N═N— group. In an embodiment, for example,an azo group of a compound of the invention includes a cyclic groupwherein a carbon-carbon bond in a carbocyclic or heterocyclic ring isreplaced with a nitrogen-nitrogen double bond (i.e. N═N). In anembodiment, for example, an azo compound of the invention includes afused ring structure comprising one or more aromatic groups and one ormore alicyclic groups, wherein a carbon-carbon bond in a carbocyclic orheterocyclic ring of the alicyclic group is replaced with anitrogen-nitrogen double bond (i.e. N═N).

The term “heterocyclic” refers to ring structures containing at leastone other kind of atom, in addition to carbon, in the ring. Examples ofsuch atoms include nitrogen, oxygen and sulfur. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, piperidyl,imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl,pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl,imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl,benzothiadiazolyl, triazolyl and tetrazolyl groups.

The term carbocyclic refers to ring structures containing only carbonatoms in the ring. Carbon atoms of carbocyclic rings may be bonded to awide range of other atoms and functional groups.

Alicyclic refers to a ring that is not an aromatic ring. Alicyclic ringsinclude both carbocyclic and heterocyclic rings.

Alkoxyalkyl: As used herein, the term “alkoxyalkyl” refers to asubstituent of the formula alkyl-O-alkyl.

Polyalkoxyalkyl: As used herein, the term “polyalkoxyalkyl” refers to asubstituent of the formula alkyl-(alkoxy)_(n)-alkoxy wherein n is aninteger from 1 to 10, preferably 1 to 4, and more preferably for someembodiments 1 to 3.

As used herein, the term “luminescence” refers to the emission ofelectromagnetic radiation from excited electronic states of atoms ormolecules. Luminescence generally refers to electromagnetic radiationemission, such as photoluminescence, chemiluminescence, andelectrochemiluminescence, among others. In photoluminescence, includingfluorescence and phosphorescence, the excited electronic state iscreated by the absorption of electromagnetic radiation. Luminescencedetection involves detection of one or more properties of theluminescence or associated luminescence process. These properties mayinclude intensity, excitation and/or emission spectrum, polarization,lifetime, and energy transfer, among others. These properties may alsoinclude time-independent (steady-state) and/or time-dependent(time-resolved) properties of the luminescence. Representativeluminescence techniques include fluorescence intensity (FLINT),fluorescence polarization (FP), fluorescence resonance energy transfer(FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS),fluorescence recovery after photobleaching (FRAP), and bioluminescenceresonance energy transfer (BRET), among others. By way of example, whenan optical agent is used in the present invention, it is desirable thatthe wavelength of non-ionizing radiation be such that it excites theoptical agent. This excitation causes a bond of the molecule to breakthus releasing an appropriate radical. This excitation may also causethe molecule to emit part of the absorbed energy at a differentwavelength; such emission may be detected using fluorometric techniquesas described above. One skilled in the art can readily determine themost appropriate optional detection technique based, at least in part,the specific agent(s) administered and/or the particular use (e.g., areato be imaged).

As used herein, the term “controlled-release component” refers to anagent that facilitates the controlled-release of a compound including,but not limited to, polymers, polymer matrices, gels, permeablemembranes, liposomes, microspheres, or the like, or any combinationthereof. Methods for producing compounds in combination withcontrolled-release components are known to those of skill in the art.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of an appropriate federal or state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly in humans ordoes not impart significant deleterious or undesirable effect on asubject to whom it is administered and in the context in which it isadministered.

As is customary and well known in the art, hydrogen atoms in formulas(FX1)-(FX25) are not always explicitly shown, for example, hydrogenatoms bonded to the carbon atoms of aromatic and alicyclic rings are notalways explicitly shown in formulas (FX1)-(FX25). The structuresprovided herein, for example in the context of the description offormulas (FX1)-(FX25), are intended to convey to one of reasonable skillin the art the chemical composition of compounds of the methods andcompositions of the invention, and as will be understood by one of skillin the art, the structures provided do not indicate the specific bondangles between atoms of these compounds.

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

As to any of the above groups which contain one or more substituents, itis understood that such groups do not contain any substitution orsubstitution patterns which are sterically impractical and/orsynthetically non-feasible. In addition, the compounds of this inventioninclude all stereochemical isomers arising from the substitution ofthese compounds.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptableanions and/or cations. As used herein, the term “pharmaceuticallyacceptable salt” can refer to acid addition salts or base addition saltsof the compounds in the present disclosure. A pharmaceuticallyacceptable salt is any salt which retains at least a portion of theactivity of the parent compound and does not impart significantdeleterious or undesirable effect on a subject to whom it isadministered and in the context in which it is administered.Pharmaceutically acceptable salts include metal complexes and salts ofboth inorganic and organic acids. Pharmaceutically acceptable saltsinclude metal salts such as aluminum, calcium, iron, magnesium,manganese and complex salts. Pharmaceutically acceptable salts include,but are not limited to, acid salts such as acetic, aspartic,alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic,bicarbonic, bisulfuric, bitartaric, butyric, calcium edetate, camsylic,carbonic, chlorobenzoic, -cilexetil, citric, edetic, edisylic, estolic,esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic,glycolylarsanilic, hexamic, hexylresorcjnoic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic,lactobionic, maleic, malic, malonic, mandelic, methanesulfonic,methylnitric, methylsulfuric, mucic, muconic, napsylic, nitric, oxalic,p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogenphosphoric, dihydrogen phosphoric, phthalic, polygalactouronic,propionic, salicylic, stearic, succinic, sulfamic, sulfanlic, sulfonic,sulfuric, tannic, tartaric, teoclic, toluenesulfonic, and the like.Pharmaceutically acceptable salts may be derived from amino acids,including but not limited to cysteine. Other pharmaceutically acceptablesalts may be found, for example, in Stahl et al., Handbook ofPharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; VerlagHelvetica Chimica Acta, Zürich, 2002. (ISBN 3-906390-26-8).Pharmaceutically-acceptable cations include among others, alkali metalcations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations (e.g., Ca²⁺,Mg²⁺), non-toxic heavy metal cations and ammonium (NH₄ ⁺) andsubstituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl, orsubstituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations). Pharmaceutically-acceptable anions include amongother halides (e.g., Cl⁻, Br⁻), sulfate, acetates (e.g., acetate,trifluoroacetate), ascorbates, aspartates, benzoates, citrates, andlactate.

The compounds of this invention may contain one or more chiral centers.Accordingly, this invention is intended to include racemic mixtures,diasteromers, enantiomers, tautomers and mixtures enriched in one ormore stereoisomer. The scope of the invention as described and claimedencompasses the racemic forms of the compounds as well as the individualenantiomers and non-racemic mixtures thereof.

Before the present methods are described, it is understood that thisinvention is not limited to the particular methodology, protocols andreagents described, as these may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of theinvention which will be limited only by the appended claims.

In certain embodiments, the invention encompasses administering opticalagents useful in the invention to a patient or subject. A “patient” or“subject”, used equivalently herein, refers to an animal. In particular,an animal refers to a mammal, preferably a human. The subject mayeither: (1) have a condition diagnosable by administration of an opticalagent of the invention; or (2) is susceptible to a condition that isdiagnosable by administering an optical agent of this invention. Thepatient may also be involved in a surgical procedure for treatment of aseparate disorder.

Compositions of the invention includes formulations and preparationscomprising one or more of the present optical agents provided in anaqueous solution, such as a pharmaceutically acceptable formulation orpreparation. Optionally, compositions of the invention further compriseone or more pharmaceutically acceptable surfactants, buffers,electrolytes, salts, carriers, binders, coatings, preservatives and/orexcipients.

In an embodiment, the invention provides a pharmaceutical formulationhaving an active ingredient comprising a composition of the invention,such as a compound of any one of formulas (FX1)-(FX25). In anembodiment, the invention provides a method of synthesizing acomposition of the invention or a pharmaceutical formulation thereof,such as a compound of any one of formulas (FX1)-(FX25). In anembodiment, a pharmaceutical formulation comprises one or moreexcipients, carriers, diluents, and/or other components as would beunderstood in the art. Preferably, the components meet the standards ofthe National Formulary (“NF”), United States Pharmacopoeia (“USP”;United States Pharmacopeial Convention Inc., Rockville, Md.), orHandbook of Pharmaceutical Manufacturing Formulations (Sarfaraz K.Niazi, all volumes, ISBN: 9780849317521, ISBN 10: 0849317525; CRC Press,2004). See, e.g., United States Pharmacopeia and National Formulary (USP30-NF 25), Rockville, Md.: United States Pharmacopeial Convention; 2007;and 2008, and each of any earlier editions; The Handbook ofPharmaceutical Excipients, published jointly by the American PharmacistsAssociation and the Pharmaceutical Press (Pharmaceutical Press (2005)(ISBN-10: 0853696187, ISBN-13: 978-0853696186); Merck Index, Merck &Co., Rahway, N.J.; and Gilman et al., (eds) (1996); Goodman andGilman's: The Pharmacological Bases of Therapeutics, 8th Ed., PergamonPress. In embodiments, the formulation base of the formulations of theinvention comprises physiologically acceptable excipients, namely, atleast one binder and optionally other physiologically acceptableexcipients. Physiologically acceptable excipients are those known to beusable in the pharmaceutical technology sectors and adjacent areas,particularly, those listed in relevant pharmacopeias (e.g. DAB, Ph.Eur., BP, NF, USP), as well as other excipients whose properties do notimpair a physiological use.

In an embodiment, an effective amount of a composition of the inventionis a “diagnostically effective” amount. As used herein, the phrase“diagnostically effective” qualifies the amount of compound administeredin diagnosis, for example of a disease state or other pathologicalcondition. The amount achieves the goal of being detectable whileavoiding adverse side effects found with higher doses. In an embodiment,an active ingredient or other component is included in a therapeuticallyacceptable amount. In an embodiment, an active ingredient or othercomponent is included in a diagnostically acceptable amount.

Variations on compositions including salts and ester forms of compounds:Compounds of this invention and compounds useful in the methods of thisinvention include those of the compounds and formula (s) describedherein and pharmaceutically-acceptable salts and esters of thosecompounds. In embodiments, salts include any salts derived from theacids of the formulas herein which acceptable for use in human orveterinary applications. In embodiments, the term esters refers tohydrolyzable esters of compounds of the names and structural formulasherein. In embodiments, salts and esters of the compounds of theformulas herein can include those which have the same or bettertherapeutic, diagnostic, or pharmaceutical (human or veterinary) generalproperties as the compounds of the formulas herein. In an embodiment, acomposition of the invention is a compound or salt or ester thereofsuitable for pharmaceutical formulations.

In an embodiment, the invention provides a method for diagnosing amedical condition comprising administering to a subject (e.g. patient)in need thereof, a diagnostically effective amount of a composition ofthe invention, such as a compound of any one of formulas (FX1)-(FX25).In an embodiment, the medical condition is acute renal failure orvarious other diseases, injuries, and disorders, including renal systemdisorders such as declining renal function, liver failure, renalfailure, and failure of one or more organs or aspects of the renalsystem.

In an embodiment, the invention provides a medicament which comprises adiagnostically effective amount of one or more compositions of theinvention. In an embodiment, the invention provides a method for makinga medicament for diagnosis or aiding in the diagnosis of a conditiondescribed herein. In an embodiment, the invention provides the use ofone or more compositions set forth herein for the making of amedicament.

In accordance with the present invention, one protocol for assessingphysiological function of body cells includes administering an effectiveamount of a compound represented by Formula (FX1) to a patient. Anappropriate dosage of the compound that is administered to a patient isreadily determinable by one of ordinary skill in the art and may varyaccording to the clinical procedure contemplated, generally ranging fromabout 1 nanomolar to about 100 micromolar. The administration of thecompound to the patient may occur in any of a number of appropriatefashions including, but not limited to: (1) intravenous,intraperitoneal, or subcutaneous injection or infusion; (2) oraladministration; (3) transdermal absorption through the skin; and (4)inhalation.

Compounds of this invention may be administered as solutions in mostpharmaceutically acceptable intravenous carriers known in the art.Pharmaceutically acceptable carriers that are well known to thoseskilled in the art include, but are not limited to, 0.01-0.1 M phosphatebuffer or 0.8% saline. Additionally, pharmaceutically acceptablecarriers may be aqueous or non-aqueous solutions, suspensions,emulsions, or appropriate combinations thereof. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Examples of aqueous carriers are water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Exemplaryparenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils. Exemplaryintravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases, and the like.

Suitable diluents, preservatives, solubilizers, emulsifiers, adjuvantand/or excipients are also suitable carriers. Such carriers are liquidsor lyophilized or otherwise dried formulations and include diluents ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), complexationwith metal ions, or incorporation of the material into or ontoparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such carriers may influence the physical state,solubility, stability, rate of in vivo release, and/or rate of in vivoclearance.

Still referring to the above-mentioned protocol, the compound may beexposed to visible and/or near infrared light. This exposure of thecompound to light may occur at any appropriate time but preferablyoccurs while the compound is located in the body (e.g., within thebloodstream and/or urinary system). Due to this exposure of the compoundto the visible and/or near infrared light, the compound luminesces,emitting spectral energy (e.g., visible and/or near infrared light) thatmay be detected by appropriate detection equipment. The luminescencefrom the compound tends to exhibit a wavelength range greater than awavelength range absorbed by the compound during excitation. Forexample, if an embodiment of the compound absorbs light of about 700 nm,the compound may luminesce, emitting light of about 745 nm.

Detection of the compound (or more particularly, the luminescencetherefrom) may be achieved through optical fluorescence, absorbance orlight scattering procedures known in the art. In one embodiment, thisdetection of the luminesced spectral energy may be characterized as acollection of the luminesced spectral energy and a generation ofelectrical signal indicative of the collected spectral energy. Themechanism(s) utilized to detect the luminescence from the compound thatis present in the body may be designed to detect only selectedwavelengths (or wavelength ranges) and/or may include one or moreappropriate spectral filters. Various catheters, endoscopes, ear clips,hand bands, head bands, surface coils, finger probes and the like may beutilized to expose the compound to light and/or to detect the lightluminesced therefrom [30]. This detection of luminescence may beaccomplished at one or more times intermittently or may be substantiallycontinuous.

Compounds of this invention may be provided in the form of a kitcomprising a compound packaged in a container. In some embodiments, thecompound may be dissolved a pharmaceutically acceptable carrier andprovided in a single container. The pharmaceutically acceptable carriermay comprise any suitable vehicle, diluent, preservative, solubilizer,emulsifier, adjuvant, excipient and/or the like such as is known in theart and/or as is described above. In some embodiments, the compound maybe in one container (e.g., in a dried or lyophilized form), and thepharmaceutically acceptable carrier may be in a separate container, allof which are packaged together in the kit. Kits of the invention mayalso include a package insert providing instructions for use.

Renal function of a patient may be determined based on the detectedluminescence. This may be achieved by using data indicative of thedetected luminescence and generating an intensity/time profileindicative of a clearance of the compound from the body. This profilemay be correlated to a physiological or pathological condition. Forexample, the patient's clearance profiles and/or clearance rates may becompared to known clearance profiles and/or rates to assess thepatient's renal function and to diagnose the patient's physiologicalcondition. In the case of analyzing the presence of the compound inbodily fluids, concentration/time curves may be generated and analyzed(preferably in real time) using an appropriate microprocessor todiagnose renal function.

Physiological function may be assessed by: (1) comparing differences inmanners in which normal and impaired cells and/or tissues remove acompound of the invention from the bloodstream; (2) measuring a rate oran accumulation of a compound of the invention in organs or tissues;and/or (3) obtaining tomographic images of organs or tissues having acompound of the invention associated therewith.

The invention is further detailed in the following Examples, which areoffered by way of illustration and are not intended to limit the scopeof the invention in any manner.

General Experimental Conditions: Unless otherwise noted, all reagentswere used as supplied. Analytical TLC was performed on Analtech silicagel GF plates (250 mm). Flash chromatography was carried out usingeither EMD Chemicals Inc. silica gel 60 (40-63 mm) or RediSep pre-packedsilica gel columns on CombiFlash chromatography system. RP-LC/MS (ESI,positive ion mode) analyses were carried out on a Waters Micromass ZQsystem equipped with a PDA detector using ThermoElectron Hypersil GoldC18 3 μm (4.6 mm×50 mm) column (gradient: 5-95% B/6 min; flow rate: 1ml; mobile phase A: 0.05% TFA in H₂O; mobile phase B: 0.05% TFA inCH₃CN). Preparative RP-HPLC was carried out using a Waters Dual Pumpsystem equipped with a Liquid Handler and a PDA detector [column: WatersXBrdige™ Prep C18 OBD™ 5 μm 30×150 mm or Sunfire™ Prep C18 5 μm OBD™30×150 mm; λ_(max): PDA (200-600 nm); flow rate: 50 mL/min; gradient:5-20 to 40-95% B/10-15 min; mobile phase A: 0.1% TFA in H₂O; mobilephase B: 0.1% TFA in CH₃CN]. RP-HPLC analyses were carried out onAgilent 1200 series system equipped with a UV detector (column:Phenomenex Luna 5 μm C18(2) 100 Å 250×4.6 mm; flow rate: 1 mL/min;mobile phase A: 0.1% TFA in H₂O; mobile phase B: 0.1% TFA in CH₃CN). NMRspectra were recorded on either a Varian Gemini-300 or a VNMRS-500spectrometer. Chemical shifts are expressed in parts per million (δ)relative to TMS (δ=0) as an internal standard and coupling constants (J)are reported in Hz. HRMS (ESI) data was obtained on a Thermo ScientificLTQ-Orbitrap mass spectrometer equipped with an IonMax electrosprayionization source in FTMS (Fourier Transform) mode with resolution≧30K.

Example 13,6-Bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide(1)

Step 1. Synthesis of3,6-diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide(2)

To a colorless solution of m-dPEG™₁₂ amine (5.37 g, 9.60 mmol) inanhydrous DMF (100 mL), 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.793g, 4.00 mmol) was added, and the brick-red suspension was stirred atice-bath temperature in an atmosphere of N₂. Then PyBOP (5.00 g, 9.60mmol) and DIPEA (5.00 mL, 28.7 mmol) were added and somewhat lighterbrownish yellow suspension was slowly allowed to warm to roomtemperature. The reaction became almost homogeneous within an hour or soand was stirred overnight under N₂. Most of the DMF was removed underhigh vacuum; the brownish yellow syrup was first filtered throughSepadex G-10 (130 g) using water followed by chromatography over C18silica gel (YMC, 130 g) using H₂O—CH₃CN (3:1 to 3:2, v/v) as eluant. Theproduct containing yellow fractions were combined, concentrated todryness, and further dried under high vacuum to give the semi-purebis-amide 2 (5.12 g, 100%) as a brownish yellow sticky solid: RP-LC/MS(ESI) m/z 1281.9 (M+H)⁺ (t_(R)=3.76 min).

Step 2

To an orange solution of the above bis-amide 2 (2.24 g, 1.75 mmol) inanhydrous 1,2-dichloroethane (DCE, 65 mL), a solution of m-dPEG™₁₂propionaldehyde (3.00 g, 5.24 mmol) in DCE (5 mL) was added, and thereaction flask was immersed in an ice bath. Then glacial HOAc (0.30 mL,5.20 mmol) was added followed by the addition of sodiumtriacetoxyborohydride (1.11 g, 5.24 mmol) in small portions over a 30min period. The resulting reddish suspension was slowly allowed to warmto room temperature and stirred overnight (ca. 16 h) in an atmosphere ofargon. The reaction was quenched by a slow addition of saturated NaHCO₃(50 mL) at 0° C. The biphasic mixture was stirred for 30 min, layerswere separated, and the aqueous phase was further extracted with CHCl₃(2×50 mL). The combined organic extracts were washed with 50% aqueousNaCl (50 mL) and brine (50 mL) and then dried over Na₂SO₄. Removal ofsolvents gave 6.89 of red solid, which was subjected to purification bypreparative RP-HPLC (XBridge, 20-45% B/15 min). The pure fractions wereconcentrated in vacuo, the residue was co-evaporated with CH₃CN (3×50mL), and then dried under high vacuum to a constant weight to afford 1(2.57 g, 61%) as red semi-solid (solidified upon storage in therefrigerator): ¹H NMR (DMSO-d₆) 8.41 (t, 2, J=5.9), 7.30 (broad, 2),3.65-3.34 [m, 192, includes broad peak at δ 3.49 for —(CH₂CH₂O)_(n)—],3.23 (s, 12), 1.79-1.74 (quintet, 4); RP-HPLC (280 nm) 98% (t_(R)=14.73min); RP-LC/MS (ESI) m/z 1198.0 (M+2H)²⁺, 1207.2 (M+H+NH4)²⁺, 1209.2(M+H+Na)²⁺ (t_(R)=4.64 min). HRMS (ESI) m/z calculated forC₁₀₈H₂₁₂N₆O₅₀Na₃ (M+3Na)³⁺ 820.7969, found 820.7993; calculated forC₁₀₈H₂₁₂N₆O₅₀Na₂ (M+2Na)²⁺ 1219.7008, found 1219.7040; calculated forC₁₀₈H₂₁₂N₆O₅₀Na (M+Na)⁺ 2416.4123, found 2416.4162.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 23,6-Bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide(3)

Step 1. Synthesis of3,6-diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide(4)

To a mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.316 g, 1.60mmol), m-dPEG™₂₄ amine (4.00 g, 3.68 mmol), and PyBOP (1.92 g, 3.69mmol) in anhydrous DMF (120 mL), was added Et₃N (8.00 mL, 57.4 mmol) andthe reaction mixture was stirred overnight (ca. 20 h) at roomtemperature under argon. Most of the DMF was removed under high vacuum,the oil dissolved in CHCl₃ (250 mL), and washed successively with 0.50 MKHSO₄-brine (1:1, v/v), saturated NaHCO₃-brine (1:1, v/v), and brine (75mL portions). The organic phase was dried over Na₂SO₄, concentrated invacuo, and the was filtered through a bed of C18 silica gel usingCH₃CN—H₂O (3:1, v/v) as eluant to give the crude bis-amide 4 (5.32 g,142%) as a brownish yellow solid, which was used as such in the nextreaction without any further purification: RP-LC/MS (ESI) m/z 780.7(M+3H)³⁺, 1179.1 (M+H+NH₄)²⁺, 1182.0 (M+H+Na)²⁺ (t_(R)=3.88 min).

Step 2

To a brownish yellow solution of the above bis-amide 4 (3.86 g crude,1.16 mmol) in anhydrous DCE (45 mL), a solution of m-dPEG™₁₂propionaldehyde (2.00 g, 3.49 mmol) in DCE (5 mL) was added, and thereaction flask was immersed in an ice bath. Then glacial HOAc (0.20 mL,3.47 mmol) was added followed by the addition of sodiumtriacetoxyborohydride (0.740 g, 3.49 mmol) in small portions over aperiod 1 h. The resulting reddish suspension was slowly allowed to warmto room temperature and stirred overnight (ca. 22 h) in an atmosphere ofargon. The reaction was quenched by a slow addition of saturated NaHCO₃(50 mL) at 0° C. The biphasic mixture was stirred for 30 min, layerswere separated, and the aqueous phase was further extracted with CHCl₃(2×50 mL). The combined organic extracts were washed with brine (50 mL)and then dried over Na₂SO₄. Removal of solvents gave 5.93 g of redviscous oil, which was dialyzed against water using SpectraPor 7dialysis tubing (MWCO 2,000 Da) to give 2.47 g of semi-pure product thatwas subjected to further purification by preparative RP-HPLC (XBridge,20-40% B/14 min). The pure fractions were concentrated in vacuo, theresidue was co-evaporated with anhydrous EtOH (2×25 mL), and then driedunder high vacuum to a constant weight to give 3 (1.84 g, 46%) as redsemi-solid (solidified upon storage in the refrigerator): ¹H NMR(DMSO-d₆) 8.41 (t, 2, J=5.7), 7.87 (broad t, 2), 3.74-3.39 [m, 200,includes broad singlet at δ 3.49 for —(CH₂CH₂O)_(n)—], 3.34 (s, 88),3.23, 3.22 (2 s, 12), 1.86-1.72 (quintet, 4); RP-HPLC (280 nm) 97%(t_(R)=14.69 min); RP-LC/MS (ESI) m/z 1166.73 (M+H+2Na)³⁺, 1737.73(M+H+Na)²⁺, 1209.2 (M+H+Na)²⁺ (t_(R)=19.13 min, Phenomenex). HRMS (ESI)m/z calculated for C₁₅₆H₃₀₈N₆O₇₄Na₃ (M+3Na)³⁺ 1173.0066, found1173.0101; calculated for C₁₅₆H₃₀₈N₆O₇₄Na₂ (M+2Na)²⁺ 1748.0153, found1748.0199; calculated for C₁₅₆H₃₀₈N₆O₇₄Na (M+Na)⁺ 3473.0415, found3473.0421.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 33,6-bis(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide(5)

The reaction of3,6-Diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide4 (2.55 g crude, 136% yield material, 1.16 mmol) with m-dPEG™₂₄propionaldehyde (2.60 g, 2.36 mmol) in the presence of HOAc (0.20 mL,2.38 mmol) and sodium triacetoxyborohydride (0.508 g, 2.40 mmol) in DCE(40 mL) was carried out overnight as described in the preparation ofExample 2 (incomplete by RP-LC/MS analysis). At this stage, the reactionmixture was treated with more m-dPEG™₂₄ propionaldehyde (0.400 g, 0.363mmol), HOAc (0.021 mL, 0.363 mmol), and sodium triacetoxyborohydride(0.077 g, 0.363 mmol) as described above, and the reaction was continuedovernight (still incomplete). After the usual work up described inExample 2, the crude product (5.70 g) obtained was subjected topurification by preparative RP-HPLC (XBridge, 20-40% B/13 min) to afford5 (0.869 g, 24%) as a brick-red solid: ¹H NMR (DMSO-d₆) 8.42 (t, 2,J=5.8), 7.95 (broad, 2), 3.63-3.42 [m, 384, broad s at δ 3.51 for—(CH₂CH₂O)_(n)—], 3.24 (s, 12), 1.80-1.75 (quintet, 4); RP-HPLC (280 nm)96% (t_(R)=13.16 min). HRMS (ESI) m/z calculated for C₂₀₄H₄₀₄N₆O₉₈Na₄(M+4Na)⁴⁺ 1149.6596, found 1149.6617.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 43,6-bis(2,5,8,11,14,17,20,23-octaoxahexacosan-26-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide(6)

The reaction of3,6-Diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide4 (1.45 g crude, 142% yield material, 0.437 mmol) with m-dPEG™₈propionaldehyde (0.520 g, 1.31 mmol) in the presence of HOAc (0.070 mL,1.21 mmol) and sodium triacetoxyborohydride (0.280 g, 1.32 mmol) in DCE(25 mL) was carried out overnight (ca. 18 h) as described in thepreparation of Example 2 (incomplete by RP-LC/MS analysis). At thisstage, the reaction mixture was treated with more m-dPEG™₈propionaldehyde (0.230 g, 0.580 mmol), HOAc (0.070 mL, 1.21 mmol), andsodium triacetoxyborohydride (0.120 g, 0.566 mmol) as described above,and the reaction was continued overnight (completed reaction). After theusual work up described in Example 2, the crude product (2.03 g) wassubjected to purification by preparative RP-HPLC (XBridge, 20-40% B/13min) to give brick-red 6 (0.625 g, 46%): ¹H NMR (DMSO-d₆) 8.42 (t, 2,J=6.0), 7.90 (broad, 2) 3.67-3.40 [m, 256, includes broad peak at δ 3.50for —(CH₂CH₂O)_(n)—], 3.24, 3.23 (2 s, 12), 1.80-1.75 (quintet, 4);RP-HPLC (280 nm) 99% (t_(R)=14.72 min); RP-LC/MS (ESI) m/z 1033.7(M+3H)³⁺, 1559.3 (M+H+NH₄)²⁺ (t_(R)=4.08 min). HRMS (ESI) m/z calculatedfor C₁₄₀H₂₇₆N₆O₆₆Na₂ (M+2Na)²⁺ 1571.9105, found 1571.9145.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 53,6-Bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide(7)

The reaction of3,6-Diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)pyrazine-2,5-dicarboxamide4 (1.80 g purified, 0.77 mmol) with m-dPEG™₄ propionaldehyde (0.500 g,2.27 mmol) in the presence of HOAc (1.00 mL, 17.3 mmol) and sodiumtriacetoxyborohydride (0.481 g, 2.27 mmol) in DCE (100 mL) was carriedout overnight as described in the preparation of Example 2 (incompleteby RP-LC/MS analysis). At this stage, the reaction mixture was treatedwith a fresh batch of similar quantities of the reagents as describedabove and continued overnight (still incomplete). After the usual workup described in Example 2, the crude product was subjected topreparative RP-HPLC to afford 7 (0.620 g, 29%): ¹H NMR (CDCl₃)characteristic broad s at δ 3.65 and singlets at δ 3.39 and 3.38 forpoly(ethylene glycol) moieties; RP-HPLC (280 nm) 94% (t_(R)=15.36 min);RP-LC/MS (ESI) m/z 2747.2 (M+H)⁺ (t_(R)=4.83 min).

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 63,6-Bis(2,5,8,11,14,17,20,23-octaoxahexacosan-26-ylamino)-N²,N⁵-bis[(R)-1-carboxy-2-hydroxyethyl]pyrazine-2,5-dicarboxamide(8)

Step 1. Synthesis of3,6-diamino-N²,N⁵-bis[(R)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl]pyrazine-2,5-dicarboxamide(9)

A 1-L round-bottom flask equipped with a Claisen adapter and an additionfunnel was charged with D-serine benzyl ester hydrochloride (24.33 g,105 mmol) and anhydrous DMF was cannulated into it. The resultingcolorless solution was cooled in an ice-bath and stirred for 15 min inan atmosphere of N₂. Then DIPEA (19.16 mL, 110 mmol) was added dropwisevia addition funnel over a 30 min period, and after 15 min, the coolingbath was removed and, 6-diaminopyrazine-2,5-dicarboxylic acid (9.91 g,50.0 mmol) was added in one portion. The brick-red suspension wasallowed to stir for 15 min before the addition of HOBt.H₂O (17.61 g, 115mmol) in one portion. After another 15 min, the reaction flask was onceagain cooled in an ice-bath, and EDC.HCl (22.05 g, 115 mmol) was addedin portions over a 10 min period. The resulting somewhat lighter andbrown suspension was slowly allowed to warm to room temperature andstirred overnight (ca. 14 h) under N₂. The dark solution wasconcentrated to a syrupy residue under high vacuum (bath temp 60° C.)that was partitioned between EtOAc and milli-Q H₂O (400 mL each). Thelayers were separated and the aq layer was further extracted with EtOAc(3×200 mL) and the combined EtOAc extracts were successively washed with0.50 M KHSO₄, saturated NaHCO₃, H₂O, and brine (250 mL each). The dried(Na₂SO₄) extracts were filtered (Whatman) and evaporated in vacuo toleave an orange slush that was dried under high vacuum over the weekendto 23.7 g of an orange solid. The crude product was subjected to flashchromatography over silica gel (EMD 60) using CHCl₃ to CHCl₃-MeOH (97:3,v/v) as eluant in a gradient fashion (sample adsorbed on silica gel wasloaded on the column; carried out in 4 runs) to give the bis-amide 9(19.6 g, 71%) as orange solid: ¹H NMR (DMSO-d₆) 8.56 (d, 2, J=8.0 Hz),7.40-7.33 (m, 10), 6.76 (s, 4), 5.37 (t, 2, J=5.5), 5.20 (distorted ABpair, 4), 4.66-4.63 (dt, 2, J=8.0, 4.0 Hz), 3.97-3.93 (m, 2), 3.81-3.77(m, 2); ¹³C NMR (DMSO-d₆) 170.57, 165.39, 146.90, 136.33, 128.88,128.48, 128.11, 126.34, 66.70, 61.59, 54.92; RP-LC/MS (ESI) m/z 553.3(M+H)₊ (t_(R)=4.44 min). HRMS (ESI) m/z calculated for C₂₆H₂₉N₆O₈ (M+H)⁺575.1861, found 575.1859.

Step 2. Synthesis of3,6-bis(2,5,8,11,14,17,20,23-octaoxahexacosan-26-ylamino)-N²,N⁵-bis[(R)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl]pyrazine-2,5-dicarboxamide(10)

A 50 mL round-bottom flask equipped with magnetic stir bar and argoninlet was charged with the above bis-amide 9 (0.350 g, 0.633 mmol),m-dPEG™₈ propionaldehyde (1.00 g, 2.52 mmol) HOAc (0.190 g, 3.14 mmol)in DCE (25 mL) and sodium triacetoxyborohydride (0.537 g, 2.53 mmol) wasadded and stirred at room temperature over the weekend. The reaction wasstirred for 30 min with saturated NaHCO₃ (25 mL), diluted with 50 mlCHCl₃ (50 mL), and the organic phase was separated and washed withbrine. The organics were dried and concentrated in vacuo to give 1.30 gof red liquid that was subjected to preparative HPLC. The pure fractionswere concentrated in vacuo to ½ volume and poured into a mixture ofsaturated NaHCO₃ and brine and extracted with CHCl3 (2×150 mL). Thecombined organics were dried and concentrated and vacuum dried to affordbis-benzyl ester 10 (0.390 g, 47%) as red oil: HRMS (ESI) m/z calculatedfor C₆₂H₁₀₁N₆O₂₄ (M+H)⁺ 1313.6862, found 1313.6907.

Step 3

A 250 mL round-bottom flask equipped with magnetic stir bar was chargedwith the above bis-benzyl ester 10 (0.390 g, 0.297 mmol) and ammoniumformate (0.112 g, 1.78 mmol) in MeOH (10 mL) and water (10 mL). To thiswas added slurry of 10% Pd/C (0.095 g) in water (10 mL) and reactionmixture was stirred at 60° C. RP-LC/MS analysis after 1 h showed ˜60%mono-ester and the rest starting material. At this stage, additionalammonium formate (0.112 g, 1.78 mmol) and 10% Pd/C (0.095 g) were addedand the reaction was completed in an hour. The catalyst was removed byfiltration through Celite, washed with deionized water (˜100 mL), andthe combined filtrates were concentrated in vacuo. The crude product waspurified by preparative HPLC and the product was concentrated in vacuoand transferred into a 4 dram vial with acetonitrile, concentrated andvacuum dried to afford 8 (0.300 g, 76%) as red oil: HRMS (ESI) m/zcalculated for C₄₈H₈₉N₆O₂₄ (M+H)⁺ 1133.5923, found 1133.5958.

The general synthetic scheme is shown in FIG. 3, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 73,6-Bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-bis[(R)-1-carboxy-2-hydroxyethyl]pyrazine-2,5-dicarboxamide(11)

Step 1.3,6-bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-bis[(R)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl]pyrazine-2,5-dicarboxamide(12)

A 100 mL round-bottom flask equipped with magnetic stir bar and argoninlet was charged with3,6-diamino-N²,N⁵-bis[(R)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl]pyrazine-2,5-dicarboxamide9 (0.800 g, 1.45 mmol), m-dPEG™₄ propionaldehyde (1.00 g, 4.54 mmol),sodium triacetoxyborohydride (0.962 g, 4.54 mmol) in DCE (25 mL) andHOAc (0.50 mL, 8.67 mmol) was added and stirred at room temperatureovernight (RP-LC/MS: ˜50% reaction). The reaction mixture was treatedwith more of m-dPEG™₄ propionaldehyde (0.508 g, 2.31 mmol), sodiumtriacetoxyborohydride (2.24 mmol), and HOAc (0.50 mL, 8.67 mmol), andthen stirred overnight under argon (still incomplete). The reactionmixture was diluted with CH₂Cl₂ (200 mL) and washed with saturatedNaHCO₃ (2×200 mL). The organic layer was dried over Na₂SO₄, the solventswere removed in vacuo, and the residue was subjected to preparativeRP-HPLC to afford the bis-benzyl ester 12 (0.501 g, 36%).

Step 2

A 50 mL round-bottom flask equipped with magnetic stir bar was chargedwith bis-benzyl ester 12 (0.501 g, 0.521 mmol), dissolved in EtOH—H₂O(20 mL; 3:1, v/v), and 10% Pd—C (100 mg) was added as slurry in water(1-2 mL). The reaction mixture was purged thoroughly with argon and thenwith H₂ and stirred at room temperature for 2 h in an atmosphere of H₂.The catalyst was removed by filtration, solvents were removed in vacuo,and the crude product subjected to purification by preparative RP-HPLCto give 11 (0.108 g, 26%).

The general synthetic scheme is shown in FIG. 3, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 83,6-Bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-bis(2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide(13)

Step 1.3,6-bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]pyrazine-2,5-dicarboxamide(14)

To a yellow suspension of3,6-diamino-N²,N⁵-bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]pyrazine-2,5-dicarboxamideof (0.424 g, 1.00 mmol) in anhydrous DCE (25 mL), m-dPEG™₄propionaldehyde (0.661 g, 3.00 mmol) was added, and the reaction flaskwas immersed in an ice bath. Then HOAc (0.17 mL, 2.95 mmol) was addedfollowed by the addition of sodium triacetoxyborohydride (0.628 g, 2.96mmol) in small portions over a 40 min period. The resulting reddishsuspension was slowly allowed to warm to room temperature and stirredovernight (ca. 20 h) in an atmosphere of argon (incomplete by RP-LC/MSanalysis). The reaction mixture was treated with more m-dPEG™₄propionaldehyde (0.165 g, 0.749 mmol), HOAc (0.040 mL, 0.694 mmol), andsodium triacetoxyborohydride (0.160 g, 0.755 mmol) as described above,and after 8 h, quenched by slow addition of saturated NaHCO₃ (50 mL) at0° C. The compound was extracted into CHCl₃ (150 mL), the organic layerwas washed with water (50 mL) followed by brine (50 mL), and then driedover Na₂SO₄. Removal of solvents gave diacetonide 14 (1.15 g) as areddish gum that was used as such in the next reaction: RP-LC/MS (ESI)m/z 834.4 (M+H)⁺ (t_(R)=4.53 min).

Step 2

To a reddish solution of the above dicetonide 14 (1.00 mmol) in THF (25mL), was added 1.0 N HCl (5 mL) and stirred for 5 h in an atmosphere ofargon. Most of the THF was removed from the reaction mixture,neutralized by 1.0 N NaOH, and concentrated in vacuo. The crude product(2.40 g) was subjected to preparative RP-HPLC (XBridge, 10-50% B/12 min)to afford diastereomeric 13 (0.554 g, 74%) as red gum: UV (λ_(max)) 499nm; ¹H NMR (CDCl₃) 8.66, 8.56 (2 t, 2), 6.24 (broad s, 6), 4.40 (t, 1.5,J=4.2, 5.2), 4.23-4.15 (m, 1.5), 3.97-3.90 (m, 3), 3.70-3.50 (m, 36),3.37, 3.36 (2 s, 6), 1.98-1.90 (quintet, 4); RP-HPLC (254 nm) 98%(t_(R)=17.43 min, 5-50% B); RP-LC/MS (ESI) m/z 754.5 (M+H)⁺ (t_(R)=3.58min). HRMS (ESI) m/z calculated for C₃₂H₅₁N₆O₁₄ (M+H)⁺ 753.4240, found753.4252.

The general synthetic scheme is shown in FIG. 4, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 93,6-Bis(2,5,8,11-tetraoxatetradecan-14-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide(15)

The reaction of3,6-diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide2 (1.00 g, 0.78 mmol) with m-dPEG™₄ propionaldehyde (0.52 g, 2.36 mmol)in the presence of HOAc (0.13 mL, 2.27 mmol) and sodiumtriacetoxyborohydride (0.50 g, 2.36 mmol) in anhyd DCE (20 mL) wascarried out overnight (ca. 18 h) as described in the preparation ofExample 1 (incomplete by RP-LC/MS analysis). At this stage, the reactionmixture was treated with more m-dPEG™₄ propionaldehyde (0.17 g, 0.77mmol), HOAc (0.130 mL, 2.27 mmol), and sodium triacetoxyborohydride(0.17 g, 0.80 mmol) as described above, and the reaction was continuedovernight (completed reaction). After the work up described in Example1, the crude product (2.22 g) was subjected to purification bypreparative RP-HPLC (XBrdige, 30-40% B/25 min) to give compound 15(0.391 g, 30%) as a red oil: ¹H NMR (DMSO-d₆) 8.42 (t, 2, J=6.0),3.40-3.55 [m, 130, includes broad peaks at δ 3.50 for —(CH₂CH₂O)_(n)—],3.24, 3.22 (2 s, 12), 1.80-1.75 (quintet, 4); RP-HPLC (280 nm) 96%(t_(R)=15.37 min); RP-LC/MS (ESI) m/z 846.2 (M+2H)²⁺, 1690.9 (M+H)⁺(t_(R)=4.07 min). HRMS (ESI) m/z calculated for C₇₆H₁₄₉N₆O₃₄ (M+H)⁺1690.0109, found 1690.0182; calculated for C₇₆H₁₄₆N₆O₃₄Na (M+Na)⁺1711.9989, found 1711.9929.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 103,6-Bis(2,6,8,11,14,17,20,23-octaoxahexacosan-26-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide(16)

The reaction of3,6-diamino-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide2 (1.00 g, 0.78 mmol) with m-dPEG™₈ propionaldehyde (0.93 g, 2.35 mmol)in the presence of HOAc (0.13 mL, 2.27 mmol) and sodiumtriacetoxyborohydride (0.50 g, 2.36 mmol) in anhyd DCE (20 mL) wascarried out overnight (ca. 18 h) as described in the preparation ofExample 1 (incomplete by RP-LC/MS analysis). At this stage, the reactionmixture was treated with m-dPEG™8 propionaldehyde (0.31 g, 0.78 mmol),HOAc (0.13 mL, 2.27 mmol), and sodium triacetoxyborohydride (0.17 g,0.80 mmol) as described above, and the reaction was continued overnight(completed reaction). After the work up described in Example 1, thecrude product (2.41 g) was subjected to purification by preparativeRP-HPLC (XBrdige, 30-40% B/20 min) to give compound 16 (0.237 g, 15%) asa red oil: ¹H NMR (DMSO-d₆) 8.41 (t, 2, J=6.0), 3.40-3.55 (m, 162,includes broad peaks at δ 3.50 for —(CH₂CH₂O)_(n)—], 3.230, 3.227 (2 s,12), 1.79-1.74 (quintet, 4); RP-HPLC (280 nm) 92% (t_(R)=13.96 min);RP-LC/MS (ESI) m/z 1022.2 (M+2H)²⁺ (t_(R)=4.09 min); HRMS (ESI) m/zcalculated for C₉₂H₁₈₂N₆O₄₂ (M+2H)²⁺ 1021.6140, found 1021.5850;calculated for C₉₂H₁₈₁N₆O₄₂ (M+H)⁺ 2042.2206, found 2042.2082;calculated for C₉₂H₁₈₀N₆O₄₂Na (M+Na)⁺ 2064.2026, found 2064.30.

The general synthetic scheme is shown in FIG. 2, where the “x” variablesrepresent various repeating units as further described and shown herein.

Example 113,6-Bis(2,5,8,11-tetraoxatetradecan-14-ylamino)pyrazine-2,5-dicarboxamide(17)

To a stirring suspension of 3,6-diamino-2,5-pyrazinedicarboxamide (0.196g, 1.00 mmol) in DCE (10 mL) is added in the following order: m-dPEG™₄propionaldehyde (0.661 g, 3.00 mmol), glacial acetic acid (0.180 g, 3.00mmol), and sodium triacetoxyborohydride (0.633 g, 3.00 mmol). After theaddition, the reaction mixture is stirred overnight at ambienttemperature. The reaction mixture is diluted with methylene chloride (10mL) and quenched with saturated NaHCO₃ (5 mL). The organic layer isseparated, washed with water, dried over anhydrous sodium sulfate,filtered, and the filtrate evaporated in vacuo. The residue is purifiedusing 20 g C18 reverse phase flash chromatography column using methanolwater gradient (0 to 20% methanol over 1 h). The desired fractions arepooled and evaporated in vacuo

Example 12 Protocol for Assessing Renal Function

An example of an in vivo renal monitoring assembly 510 is shown in FIG.5 and includes a light source 512 and a data processing system 514. Thelight source 512 generally includes or is interconnected with anappropriate device for exposing at least a portion of a patient's bodyto light therefrom. Examples of appropriate devices that may beinterconnected with or be a part of the light source 512 include, butare not limited to, catheters, endoscopes, fiber optics, ear clips, handbands, head bands, forehead sensors, surface coils, and finger probes.Indeed, any of a number of devices capable of emitting visible and/ornear infrared light of the light source may be employed in the renalmonitoring assembly 510.

Still referring to FIG. 5, the data processing system 514 of the renalmonitoring assembly 510 may be any appropriate system capable ofdetecting spectral energy and processing data indicative of the spectralenergy. For instance, the data processing system 514 may include one ormore lenses (e.g., to direct and/or focus spectral energy), one or morefilters (e.g., to filter out undesired wavelengths of spectral energy),a photodiode (e.g., to collect the spectral energy and convert the sameinto electrical signal indicative of the detected spectral energy), anamplifier (e.g., to amplify electrical signal from the photodiode), anda processing unit (e.g., to process the electrical signal from thephotodiode). This data processing system 514 is optionally configured tomanipulate collected spectral data and generate an intensity/timeprofile and/or a concentration/time curve indicative of renal clearanceof a compound of the present invention from the patient 520. Indeed, thedata processing system 514 may be configured to generate appropriaterenal function data by comparing differences in manners in which normaland impaired cells remove a compound of the invention from thebloodstream, to determine a rate or an accumulation of the compound inorgans or tissues of the patient 520, and/or to provide tomographicimages of organs or tissues having the compound associated therewith.

In one protocol for determining renal function, an effective amount of acompound of the invention is administered to the patient (e.g., in theform for a pharmaceutically acceptable composition). At least a portionof the body of the patient 520 is exposed to visible and/or nearinfrared light from the light source 512 as indicated by arrow 516. Forinstance, the light from the light source 512 may be delivered via afiber optic that is affixed to an ear of the patient 520. The patientmay be exposed to the light from the light source 512 before and/orafter administration of the compound to the patient 520. In some cases,it may be beneficial to generate a background or baseline reading oflight being emitted from the body of the patient 520 (due to exposure tothe light from the light source 512) before administering the compoundto the patient 520. When the compound that is in the body of the patient520 is exposed to the light from the light source 512, the compoundluminesces, emitting light (indicated by arrow 518) that isdetected/collected by the data processing system 514. Initially,administration of the compound to the patient 520 generally enables aninitial spectral signal indicative of the initial content of thecompound in the patient 520. The spectral signal then tends to decay asa function of time as the compound is cleared from the patient 520. Thisdecay in the spectral signal as a function of time is indicative of thepatient's renal function. For example, in a first patient exhibitinghealthy/normal renal function, the spectral signal may decay back to abaseline in a time of T. However, a spectral signal indicative of asecond patient exhibiting deficient renal function may decay back to abaseline in a time of T+4 hours. As such, the patient 520 may be exposedto the light from the light source 512 for any amount of timeappropriate for providing the desired renal function data. Likewise, thedata processing system 514 may be allowed to collect/detect spectralenergy for any amount of time appropriate for providing the desiredrenal function data.

Referring now to FIG. 6, a further example of an in vivo monitoringassembly includes a source of electromagnetic radiation 610, anelectromagnetic radiation detector 670 and a data processing system 680.The electromagnetic radiation source 610 generally includes or isinterconnected with an appropriate device or devices 620 for exposing atleast a portion of a subject's body to electromagnetic radiation from630. Examples of appropriate devices 620 that may be operativelyconnected to, or be a part of, the electromagnetic radiation source 610include, but are not limited to, catheters, endoscopes, fiber optics,ear clips, hand bands, head bands, forehead sensors, surface coils, andfinger probes. Indeed, any of a number of devices capable of emittingvisible and/or near infrared electromagnetic radiation may be employedin a monitoring assembly.

The electromagnetic radiation detector 670 of the monitoring assemblymay be any appropriate system capable of collecting and detectingelectromagnetic radiation emitted from a subject 690. Theelectromagnetic radiation detector 670 may be operatively connected to,for example, one or more optical collection elements 650. The opticalcollection elements 650 of the monitoring assembly may include, amongother elements, lenses, mirrors, filters, and fiber optics.Electromagnetic radiation detectors 670 suitable for use with themonitoring assembly include, but are not limited to, CCD detectors, CMOSdetectors, photodiode detectors, photodiode array detectors, andphotomultiplier tube detectors.

The data processing system 680 of the monitoring assembly may be anyappropriate system capable of processing data obtained from theelectromagnetic radiation detector 670. For instance, the dataprocessing system 680 may include an amplifier (e.g., to amplify anelectrical signal from the detector), and a processing unit (e.g., toprocess the electrical signal from the detector). The data processingsystem 680 is preferably configured to manipulate collectedelectromagnetic radiation data and generate an intensity as a functionof time profile and/or a concentration as a function of time curveindicative of clearance of detectable agent, such as a compound of theformula (FX1)-(FX25), from a subject 690. Indeed, the data processingsystem 680 may be configured to generate appropriate data by comparingamount or concentration of the composition in the bloodstream or bodilyfluid, to determine a rate of excretion or an accumulation of thecomposition in cells, organs or tissues of the subject 690, and/or toprovide tomographic images of cells, organs or tissues having theoptically functional composition associated therewith.

In one protocol for monitoring, an effective amount of a composition,such as a compound of the formula (FX1), is administered to the subject.At least a portion of the body of the subject is exposed toelectromagnetic radiation 630 from the electromagnetic radiation source610. For instance, the electromagnetic radiation 630 from theelectromagnetic radiation source 610 may be delivered via a fiber optic620 that is affixed to an ear of the subject 690. The subject 690 may beexposed to the electromagnetic radiation 630 from the electromagneticradiation source 610 before, after or during administration of thecomposition to the subject 690. In some cases, it may be beneficial togenerate a background or baseline reading of electromagnetic radiation640 being emitted from the body of the subject 690, due to exposure tothe electromagnetic radiation 630 from the electromagnetic radiationsource 610, before administering the composition to the subject 690.When the optically functional composition that is in the body of thesubject 690 is exposed to the electromagnetic radiation 630 from theelectromagnetic radiation source 610, the optically functionalcompositions emit electromagnetic radiation 640 that is collected by theoptical collection elements 650 and detected by the electromagneticradiation detector 670. The signal from the electromagnetic radiationdetector 670 is then analyzed by the data processing system 680.

Example 13 Photophysical and Renal Clearance Properties of ExemplaryCompounds

Spectral and pharmacokinetic properties, photophysical properties, urinepercentages, tissue clearance (optical) and plasma pharmacokineticclearances of some compounds were measured according to the methodsdescribed below and herein. Table 1 shows the results of thesemeasurements.

TABLE 1 Photophysical properties, urine clearance, optical monitoringand pharmacokinetic data of compounds in Examples 1-10. PhotophysicalTissue Properties Percent found Clearance Excitation Emission inUrine^(a) (Optical) Pharmacokinetics Compound λ_(max) (nm) λ_(max) (nm)(6 hrs) T_(1/2)β^(a) (min) T_(1/2)β^(a) (min) Clearance^(a) 1 500 605   97 ± 0.4 (3)^(b) 19.9 ± 2.4 (3) 18.8 ± 0.6 (3) 3.1 ± 0.2 (3)  [18.0 ±0.3 (3)]^(c) [3.3 ± 0.1 (3)] 3 499 602 89 ± 2 (3) 20.4 ± 2.0 (4) 30.5 ±3.2 (3) 2.5 ± 0.2 (3) 5 495 603 86 ± 7 (3) 17.6 ± 0.9 (4) — — 6 498 60392 ± 4 (3) 19.1 ± 0.8 (4) — — 7 494 602 97 ± 2 (3) 14.6 ± 1.1 (4) — — 8495 605 61 ± 7 (3) — — — 13 486 607 29 ± 2 (3) — — — 15 492 603 86 ± 1(3) — — — 16 492 605 87 ± 1 (3) — — — ^(a)Given as mean ± SEM.^(b)Numbers in parentheses indicate number of test animals. ^(c)Numbersin brackets correspond to probenecid challenge experiment.

Photophysical Properties and Protein Binding.

In general, renally excretable compounds are dissolved in PBS buffer toform a 2 mM stock solution. The UV absorbance properties are determinedon a 100 μM solution in PBS using a UV-3101PC UV-Vis-NIR Scanningspectrophotometer system from Shimadzu. The fluorescence properties(λ_(ex), λ_(em), and CPS at λ_(em)) are determined on a 10 μM solutionin PBS using a Fluorolog-3 spectrofluorometer system from Jobin YvonHoriba. The percent plasma protein binding is determined on a 20 μMcompound solution in rat plasma incubated at 37° C. for 1 h. Theseparation of free from bound is made using an Amicon Centrifree YM-30device (Regenerated Cellulose 30,000 MWCO) and a Z400K RefrigeratedUniversal Centrifuge from Hermle. The concentration of protein-free isdetermined via HPLC analysis using a set of external calibrationstandards and fluorescence detection.

Urine Elimination Studies.

Rat urine elimination studies are conducted in either conscious oranesthetized Sprague-Dawley rats. The test compound (1 mL, 2 mM in PBS)is administered by tail vein injection into conscious, restrained rats,with subsequent collection of urine at the time points of 2, 4 and 6 hpost injection. The metabolic cages are washed with water to maximizethe recovery of urine discharged at each time point. Alternatively, ratsare anesthetized with 100 mg/kg Inactin intraperitoneally, a tracheatube is inserted to maintain adequate respiration, and 1 mL of testcompound is injected into the lateral tail vein. Rats are placed on 37°C. heating pad during the entire experiment. At 6 h post injection, theabdomen is opened, and the urine is removed from the bladder using a 21gauge needle and a 3 cc syringe. Quantitation of each compound in urineis performed via HPLC analysis using a set of external calibrationstandards and fluorescence detection. The percent recovery of compoundin urine at each time point is calculated based on the balance of mass.

Non-Invasive Optical Pharmacokinetic Studies.

Male Sprague-Dawley rats (330-380 g) are anesthetized by Inactin (I.P.)or 2% Isoflurane gas anesthesia delivered by a small rodent gasanesthesia machine (RC2, Vetequip, Pleasanton, Calif.). The animals areplaced on a heated board where temperature is maintained between 36-38°C. One ear lobe is glued flat to a glass slide positioned approximately2 mm beneath a fiber optic bundle for recording fluorescence from a testcompound passing through the ear. After a 100 second baseline recording,1 mL of a 2 mM solution is injected into the tail-vein of the rat andthe fluorescence signal corresponding to plasma and tissue distributionand subsequent renal clearance of the compound is monitored at the ear.The pharmacokinetic parameters of the compounds are analyzed usingWinNonLin pharmacokinetic modeling software (Pharsight, Mountain View,Calif.) and Microsoft (Redmond, Wash.) Excel. This method is used todetect renally excretable compounds of the invention and FIG. 8Aprovides a plot illustrating non-invasive in vivo fluorescence as afunction of time following delivery of a renally excretable compound.

Optical Monitoring Apparatus and Protocol.

A schematic of the apparatus for non-invasive in vivo detection offluorescence is shown in FIG. 7. A nominal 473 nm solid state lasersource (100) is employed (Power Technology model LDCU12/7314). The lasersource is directed into one leg (110) of a silica bifurcated fiber opticbundle (120) (Oriel #77565). The common end of this bifurcated bundle(130) is placed approximately 2 mm from the rat ear (140). The secondleg of the bifurcated fiber optic bundle (150) is fitted with acollimating beam probe (160) (Oriel #77644). A long pass filter (170)(Semrock LP02-488RS-25) and narrow band interference filter (180)(Semrock FF01-593/40-25) are placed in front of a photomultiplier tube(190) (Hamamatsu photosensor module H7827-011).

A chopper (200) (Stanford Research Systems model SR540) is placed afterthe laser and before the launch into the bifurcated cable. The output ofthe photosensor is connected to a lock-in amplifier (210) (StanfordResearch Systems model SR830). The lock-in output is digitized (220)(National Instruments NI-USB-6211) and the digitized data is acquired bycomputer using LabVIEW® data acquisition software (230).

Invasive Pharmacokinetic Studies.

Male Sprague-Dawley rats (330-380 g) are anesthetized by Inactin (I.P.).Rats are surgically instrumented with a trachea tube (PE-190) tofacilitate breathing and femoral artery and vein catheters (PE-50 filledwith 20 units/mL heparinized saline) for blood sampling and drugadministration respectively. After administration of 1 mL of a 2 mMsolution of agent, approximately 200 μL blood is sampled and placed intoa heparinized tube (Microtainer Brand Tube w/Lithium Heparin, BD 365971)at 0, 1, 6, 12, 18, 30, 45, 60, 90, 120 min. The concentration ofcompound in each centrifuged plasma sample is determined via HPLCanalysis using a set of external calibration standards and fluorescencedetection. The resulting pharmacokinetic parameters of the compound areanalyzed using WinNonLin pharmacokinetic modeling software (Pharsight,Mountain View, Calif.). This method is used to detect renally excretablecompounds of the invention and FIG. 8B provides a plot illustratinginvasive PK (plasma concentration) as a function of time followingdelivery of a renally excretable compound.

Probenecid Inhibition Studies.

Six male Sprague-Dawley rats are treated in the same manner as describedabove in the invasive pharmacokinetic studies. These 6 rats receive 70mg/kg Probenecid (Sigma-Aldrich; St. Louis, Mo.) 10 min prior to thetest compound; this administration is flushed with 0.2 ml NaCl. Anadditional 6 rats are treated in the same fashion but do not receiveprobenecid.

Non-Invasive and Invasive Concentration Correlation.

Time course data from an invasive plasma pharmacokinetic (PK) experimentand a non-invasive optical monitoring experiment are used to correlatein vivo fluorescence with plasma concentration. By plotting the averagerelative fluorescence unit response from three optical monitoring runsversus concentration values from an invasive PK experiment for each timepoint, a good correlation can be demonstrated for a renally excretablecompound. FIG. 9 provides a plot illustrating such a linear correlation.PK parameters are determined from the optical clearance data. Glomerularfiltration rate (GFR) is estimated with reasonable accuracy from thepharmacokinetic clearance value derived from direct analysis of thenon-invasive optical monitoring data.

Example 14 Visualization of Compounds During Surgery

The compounds of any of formula (FX1)-(FX25) can be used in a surgicalprocedure to visualize bodily fluids, organs or tissues. The compoundsof any of formula (FX1)-(FX25), for example, can be administered to asubject and allowed to collect in selected bodily fluids, organs,tissues or systems, such as urine and the renal system. The surgicalarea can then be illuminated with electromagnetic radiation, forexample, electromagnetic radiation of wavelength selected over the rangeof 350 nanometers to 900 nanometers. The surgeon will then be able tosee luminescence from the administered compound should the compoundbecome exposed to the illuminating electromagnetic radiation. Forexample if a bodily fluid, such as urine, containing the administeredcompound is exposed to the illuminating electromagnetic radiation, thesurgeon will see luminescence resulting therefrom.

Once administered to a subject, the compounds of any of formula(FX1)-(FX25) allow for more successful surgical outcomes, for exampledue to the ability of a surgeon to identify those bodily fluids andorgans, such as of the renal system, which contain the administeredcompounds. Identification of these fluids and organs enables the surgeonto avoid unwanted damage to non-target organs and tissues duringsurgery. If, however, an organ or tissue comprising the administeredcompound is damaged, the luminescence from the administered compound inbodily fluids at or near the damage site will alert the surgeon to thedamage. The surgeon can then take remedial measures and thereby reducethe extent of the damage to the subject.

Example 15 Modified Pyrazine Derivatives and Uses Thereof

In an embodiment, the following compounds and methods are provided. Itis noted the variable definitions are intended to be used only in thisexample.

In a first aspect, the present invention is directed to compounds ofFormula I and their pharmaceutically acceptable salts. With regard toFormula I, each of R¹ and R³ is independently —H,—(CH₂)_(z)(CH₂CH₂O)_(a)R⁵, —(CH₂CH₂O)_(a)R⁵, —CH(COOH)CH₂OH, or C₃-C₆polyhydroxylated alkyl. Further, each of R², R⁴ and R⁵ is independently—H or C₁-C₃ alkyl. Each occurrence of ‘m’, ‘n’ and ‘z’ independentlyvaries from 3 to 6, inclusive. In addition, each occurrence of ‘p’ and‘q’ independently varies from 1 to 99, inclusive, and each occurrence of‘a’ independently varies from 1 to 100, inclusive.

With regard to Formula I, R¹ and R³ are independently —H,—(CH₂)_(z)(CH₂CH₂O)_(a)R⁵, —(CH₂CH₂O)_(a)R⁵, —CH(COOH)CH₂OH, or C₃-C₆polyhydroxylated alkyl. For instance, in some embodiments, each of R¹and R³ is —CH(COOH)CH₂OH. In other embodiments, each of R¹ and R³ is—(CH₂CH₂O)_(a)R⁵. In still other embodiments, each of R¹ and R³ is C₃-C₆polyhydroxylated alkyl (e.g., each is C₃ polyhydroxylated alkyl, or eachis C₃ or C₄ polyhydroxylated alkyl). In even other embodiments, each ofR¹ and R³ is —H. In still other embodiments, each of R¹ and R³ is—(CH₂)_(z)(CH₂CH₂O)_(a)R⁵.

Still referring to compounds of Formula I, R², R⁴ and R⁵ areindependently —H or C₁-C₃ alkyl. For example, in some embodiments, eachoccurrence of R⁵ may independently be C₁-C₃ alkyl (e.g., each occurrenceof R⁵ is C₁ alkyl). In some embodiments, each of R² and R⁴ is —H. Inother embodiments, each of R² and R⁴ is independently C₁-C₃ alkyl (e.g.,each of R² and R⁴ is C₁ alkyl). ‘m’ independently varies from 3 to 6,inclusive. For instance, in some embodiments, ‘m’ may be 3 or 4 (e.g.,‘m’ may be 3 in some embodiments). Likewise, ‘n’ independently variesfrom 3 to 6, inclusive. For instance, in some embodiments, ‘n’ may be 3or 4 (e.g., ‘n’ may be 3 in some embodiments). One of the benefits of‘m’ and ‘n’ independently varying from 3 to 6 is that the pyrazinederivative can be “tuned” to a desired wavelength. In this regard, apyrazine derivative having both ‘m’ and ‘n’ equal to 3 may absorb and/orluminesce at respective light wavelengths that are greater than (e.g.,about 10-15 nm greater than) that of a generally similar pyrazinederivative where both ‘m’ and ‘n’ are equal to 2. A similar phenomenoncould potentially be observed moving from 3 to 4, from 4 to 5, and/orfrom 5 to 6. Accordingly, pyrazine derivatives of Formula I may bedesigned to absorb and/or luminesce at light wavelengths that maypenetrate tissues better than that of lower light wavelengths.

Still referring to compounds of Formula I, each occurrence of ‘p’ and‘q’ independently varies from 1 to 99, inclusive. For instance, in someembodiments, each of ‘p’ and ‘q’ independently varies from 2 to 40,inclusive. In other embodiments, each of ‘p’ and ‘q’ independentlyvaries from 3 to 23, inclusive.

With regard to the various possibilities for R¹ and R³ in Formula I,each occurrence of ‘z’ independently varies from 3 to 6, inclusive. Forexample, each occurrence of ‘z’ may be 3 or 4 in some embodiments.Further, each occurrence of ‘a’ independently varies from 1 to 100,inclusive. For instance, in some embodiments, each occurrence of ‘a’ mayindependently vary from 10 to 40, inclusive. In other embodiments, eachoccurrence of ‘a’ independently varies from 12 to 24, inclusive.

A second aspect of the invention is directed to pharmaceuticallyacceptable compositions, each of which includes a compound (orpharmaceutically acceptable salt thereof) disclosed herein and apharmaceutically acceptable carrier.

Still a third aspect of the invention is directed to methods ofvisualizing the urinary system and determining renal function using acompound (or pharmaceutically acceptable salt thereof) of Formula I. Inthese methods, an effective amount of the compound is administered intothe body of a patient (e.g., a mammal such as a human or animalsubject). The compound in the body of the patient is exposed to visibleand/or near infrared light. Due to this exposure of the compound tolight, the compound luminesces spectral energy that may be detected byappropriate detection equipment. For example, luminescence from thecompound may be detected using an appropriate detection mechanism suchas an invasive or non-invasive optical probe. The urinary system may bevisualized and/or renal function may be determined based on theluminescence detected. For example, an initial amount of the compoundpresent in the body of a patient may be determined by amagnitude/intensity of luminescence from the compound that is detected(e.g., in the bloodstream of the patient). As the compound is clearedfrom the body, the magnitude/intensity of detected luminescence tends todiminish. Accordingly, a rate at which this magnitude of detectedluminescence diminishes may be correlated to a renal clearance rate ofthe patient. This detection may be done periodically or in substantiallyreal time (providing a substantially continuous monitoring of renalfunction). Indeed, methods of the present invention enable renalfunction/clearance to be determined via detecting one or both a changeand a rate of change of the detected magnitude of luminescence.

Example 16 Optical Imaging Using Pyrazine Compounds

In general, molecules absorbing, emitting, or scattering in the visibleor NIR region of the electromagnetic spectrum are useful for opticalmeasurement. The high sensitivity associated with fluorescence permitsdetection without the negative effects of radioactivity or ionizingradiation. Pyrazine compounds of the invention absorb strongly in thered and NIR regions. Furthermore, the electronic properties of thesesystems are very sensitive to substitution patterns in the ring of thepyrazine compound and allows for “tuning” the absorption and emissionproperties using the information described herein.

In an embodiment of this aspect, the invention provides a method ofusing an optical agent, for example, in a biomedical procedure foroptically imaging or visualizing a target tissue or a class of targettissues. The present methods include tissue selective imaging andvisualization methods, such as imaging or visualization of renal tissue.A method of this aspect comprises the step of administering adiagnostically effective amount of a compound to a subject, wherein thecompound is a compound having any of formulae (FX1) to (FX25) or apharmaceutical preparation thereof. The present methods are useful forimaging or visualizing aspects of the renal system, for example.

In methods of this aspect, the compound that has been administered tothe subject then is exposed in vivo to electromagnetic radiation andelectromagnetic radiation emitted or scattered by the compound is thendetected. In some embodiments, fluorescence is excited from the compound(e.g., due to the electromagnetic radiation exposure), optionally viamultiphoton excitation processes. In an embodiment particularly usefulfor imaging and/or visualization, the method of this aspect furthercomprises: (i) exposing a compound, such as a compound having any one offormula (FX1) to (FX25), administered to the subject to electromagneticradiation capable of exciting emission from the compound; and (ii)measuring the emission from the compound. In some embodiments, themethods of the present invention use fluorescence excitation viaexposure to light having wavelengths selected over the range of 400-1300nm. For example, optical coherence tomography (OCT) is an opticalimaging technique compatible with the present compounds that allows highresolution cross sectional imaging of tissue microstructure. OCT methodsuse wavelengths of about 1280 nm. Use of electromagnetic radiationhaving wavelengths selected over the range of 700 nanometers to 1300nanometers may be useful for some in situ optical imaging methods of thepresent invention, including biomedical applications for imaging organs,tissue and/or tumors, anatomical visualization, optical guided surgeryand endoscopic procedures. Compounds in present methods may function ascontrast agents, optical probes and/or tracer elements. The methods ofthe present invention include in vivo, in vitro and ex vivo imaging andvisualization. The present invention provides methods for a range ofclinical procedures, including optical imaging methods and/orvisualization guided surgery and/or endoscopic diagnostic andtherapeutic procedures.

The techniques utilized to detect the spectral energy from the pyrazinederivative that is present in the body may be designed to detect onlyselected wavelengths (or wavelength ranges) and/or may include one ormore appropriate spectral filters. Various catheters, endoscopes, earclips, headbands, surface coils, finger probes, and the like may beutilized to expose the pyrazine derivative to light and/or to detectlight emitting therefrom. This detection of spectral energy may beaccomplished at one or more times intermittently or may be substantiallycontinuous.

Preferably, non-ionizing energy is administered to the subject or samplefor detecting or imaging a compound of the invention. As used herein,the term “non-ionizing energy” generally refers to electromagneticradiation that does not carry enough energy to completely remove atleast one electron from an atom or molecule of the patient's body. Forexample, in some embodiments, non-ionizing energy may include spectralenergy ranging in wavelength from about 350 nm to about 1200 nm. In someembodiments, non-ionizing energy may simply include visible and/or nearinfrared light.

In one embodiment, the spectral properties of the compounds of theinvention may be tuned to desired wavelength ranges for excitationand/or emission. This may be useful, for example, in developing aparticular imaging technique using a known excitation source.

Example 17 Methods of Monitoring Organ Function Using Pyrazine Compounds

The invention provides compositions and methods for monitoring organfunction in a subject. In an embodiment, the present invention providesa method of using a detectable agent, the method comprising:

(i) administering a diagnostically effective amount of a detectableagent to a subject, for example by administering the detectable agentinto a bodily fluid of the subject, wherein the detectable agent isdifferentially separated from a bodily fluid by the organ or tissue; thedetectable agent comprising a compound having formula (FX1), or apharmaceutically acceptable salt or ester thereof:

wherein:

-   -   R¹ and R³ are each independently —H, —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵,        —CH(COOH)CH₂OH or —(CH₂)_(a)Y¹;    -   each Y¹ is independently —OR⁶, —(CHOH)_(c)R⁷, —NR⁸R⁹, —CONR⁸R⁹,        —NHCO(CHOH)_(c)R⁷ or —NHCO(CH₂)_(a)(CH₂CH₂O)_(b)R⁵;    -   each of R², R⁴, R⁵, R⁶ and R⁷ are independently —H or C₁-C₆        alkyl;    -   R⁸ and R⁹ are independently —H, C₁-C₃ alkyl,        —(CH₂)_(a)(CHOH)_(c)R⁷, or —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵;    -   each a and c is independently an integer selected from the range        of 0 to 6;    -   each b is independently an integer selected from the range of 1        to 120;    -   each p and q is independently an integer selected from the range        of 0 to 120;    -   each of m and n is independently an integer selected from the        range of 3 to 6;        (ii) exposing the detectable agent in a bodily fluid to        electromagnetic radiation for exciting emission from the        detectable agent;        (iii) measuring the emission from the detectable agent that is        in a bodily fluid; and        (iv) determining the physiological function of the organ or        tissue of the subject based on measurement of the emission. In        an embodiment, for example, the organ or tissue is a kidney, or        tissue or cells thereof, of the subject. In an embodiment, for        example, the organ or tissue is a liver, or tissue or cells        thereof, of the subject.

In an embodiment, a method of the invention of monitoring organ functioncomprises administering to a patient a compound having any one offormula selected from (FX1)-(FX25), including any of the specificcompositions classes and compounds described in connection with formula(FX1)-(FX25). As will be understood by one of skill in the art, thepresent methods of monitoring organ function expressly include methodsof using optical agents wherein the detectable agent includes thecompound classes, compounds, and all variations thereof, describedherein, including the compound classes, compounds and variationsdescribed in connection with any one of formulae (FX1)-(FX25).

In an embodiment, for example, the method further comprises exciting andmeasuring fluorescence from the detectable agent in the subject for aplurality of times after administration of the detectable agent. In anembodiment, a temporal profile of fluorescence from the detectable agentadministered to the subject is determined and evaluated with respect tocharacterizing organ functioning, for example, by measuring a rate ofchange in fluorescence (e.g., a decrease in fluorescence) as a functionof time, and optionally comparing the measured rate of change influorescence to a rate of change characteristic of a subject having ahealthy organ or a subject having a known disease condition. Organfunction can be assessed in the present methods by comparing differencesin the manner in which normal and impaired cells remove the detectableagent (also referred to as a tracer in this context) from thebloodstream, by measuring the clearance or accumulation of these tracersin the organs or tissues, and/or by obtaining tomographic images of theorgans or tissues. Blood pool clearance may be measured non-invasivelyfrom convenient surface capillaries such as those found in an ear lobeor a finger or can be measured invasively using an endovascularcatheter. Accumulation of the tracer within the cells of interest can beassessed in a similar fashion. The clearance of the tracer compounds canbe determined by selecting excitation wavelengths and filters for theemitted photons. The concentration vs time curves and/or fluorescenceintensity vs time curves may be analyzed (preferably, but notnecessarily in real time) by a microprocessor or the like.

Systems and methods of the present invention may optionally include anoptical monitoring assembly or device for detecting optical agents ofthe invention. An example of an in vivo disease state optical monitoringassembly includes a source of electromagnetic radiation, anelectromagnetic radiation detector and a data processing system. Theelectromagnetic radiation source generally includes or is interconnectedwith an appropriate device or devices for exposing at least a portion ofa patient's body to electromagnetic radiation there from. Examples ofappropriate devices that may be operatively connected to, or be a partof, the electromagnetic radiation source include, but are not limitedto, catheters, endoscopes, fiber optics, ear clips, hand bands, headbands, forehead sensors, surface coils, and finger probes. Indeed, anyof a number of devices capable of emitting visible and/or near infraredelectromagnetic radiation may be employed in an optical monitoringassembly.

The electromagnetic radiation detector of the optical monitoringassembly may be any appropriate system capable of collecting, detectingand measuring the intensity of electromagnetic radiation emitted from asubject. The electromagnetic radiation detector may be operativelyconnected to, for example, one or more optical collection elements. Theoptical collection elements of the optical monitoring assembly mayinclude, among other elements, lenses, mirrors, optical filters (e.g.,band pass filters and cut off filters), and fiber optics.Electromagnetic radiation detectors suitable for use with the diseasestate optical monitoring assembly include, but are not limited to, CCDdetectors, CMOS detectors, photodiode detectors, photodiode arraydetectors, and photomultiplier tube detectors.

The data processing system of the optical monitoring assembly may be anyappropriate system capable of processing data obtained from theelectromagnetic radiation detector. For instance, the data processingsystem may include an amplifier (e.g., to amplify an electrical signalfrom the detector), and a processing unit (e.g., to process theelectrical signal from the detector). The data processing system ispreferably configured to manipulate collected electromagnetic radiationdata and generate an intensity as a function of time profile and/or aconcentration as a function of time curve indicative of clearance of anoptical agent, conjugate, bioconjugate or integrated bioconjugatecomposition of the present invention from a subject. Indeed, the dataprocessing system may be configured to generate appropriate diseasestate or health state data by comparing differences in amount of normaland impaired cells in the bloodstream, to determine a rate or anaccumulation of the composition in cells, organs or tissues of thesubject, and/or to provide tomographic images of cells, organs ortissues having the optical agent, conjugate, bioconjugate or integratedbioconjugate composition associated therewith.

In one protocol for optical monitoring, an effective amount of acomposition having formula (FX1)-(FX25) including an optical agent,conjugate, bioconjugate or integrated bioconjugate of the invention isadministered to the subject. At least a portion of the body of thesubject is exposed to visible and/or near infrared electromagneticradiation from the electromagnetic radiation source. For instance, theelectromagnetic radiation from the electromagnetic radiation source maybe delivered via a fiber optic that is affixed to an ear of the subject.The subject may be exposed to electromagnetic radiation from theelectromagnetic radiation source before, during or after administrationof the composition to the subject. In some cases, it may be beneficialto generate a background or baseline reading of electromagneticradiation being emitted from the body of the subject, due to exposure tothe electromagnetic radiation from the electromagnetic radiation source,before administering the composition to the subject. When the opticalagents, conjugates, bioconjugates or integrated bioconjugates of thecomposition that are in the body of the subject are exposed to theelectromagnetic radiation from the electromagnetic radiation source, theoptical agents, conjugates, bioconjugates or integrated bioconjugatesemit electromagnetic radiation that is collected by optical collectionelements and detected by the electromagnetic radiation detector. Thesignal from the electromagnetic radiation detector is then analyzed bythe data processing system.

Initially, administration of the composition to the subject generallyenables an electromagnetic radiation signal indicative of the content ofthe optical agent(s), conjugate(s), bioconjugate(s) or integratedbioconjugate(s) in the subject. In some embodiments, the electromagneticradiation signal tends to decay as a function of time as the opticalagent(s), conjugate(s), bioconjugate(s) or integrated bioconjugate(s) iscleared from the subject. In a subject with a healthy disease state, theelectromagnetic radiation signal will decay to near the baseline levelas the optical agent(s), conjugate(s), bioconjugate(s) or integratedbioconjugate(s) is cleared from the subject. In a subject with anunhealthy disease condition, the optical agent(s), conjugate(s),bioconjugate(s) or integrated bioconjugate(s) will not be cleared by thesubject during the time scale of the monitoring, or will be cleared at arate which differs from the healthy disease state clearance rate. As aresult, the electromagnetic radiation signal may decay at a differentrate. Alternatively, the electromagnetic radiation signal may notdecrease to the baseline level, but will remain at an elevated level.The difference between this increased electromagnetic radiation signallevel (or decay rate) and the baseline level (or decay rate) may beindicative of a disease state in the subject. Some methods of thepresent invention further comprise comparing the rate of decay offluorescence intensity at a number of different times so as to assessthe state of organ function. As such, the subject may be exposed to theelectromagnetic radiation from the electromagnetic radiation source forany amount of time appropriate for providing the desired disease statemonitoring data. Likewise, the electromagnetic radiation collection,detection, and data processing systems may be allowed to collect anddetect electromagnetic radiation for any amount of time appropriate forproviding the desired disease state monitoring data.

In addition to noninvasive techniques, a modified pulmonary arterycatheter that can be used to make desired measurements has beendeveloped. This is a distinct improvement over current pulmonary arterycatheters that measure only intravascular pressures, cardiac output andother derived measures of blood flow. Current critically ill patientsare managed using these parameters but rely on intermittent bloodsampling and testing for assessment of renal function. These laboratoryparameters represent discontinuous data and are frequently misleading inmany patient populations. Yet, importantly, they are relied upon heavilyfor patient assessment, treatment decisions, and drug dosing.

The modified pulmonary artery catheter incorporates an optical sensorinto the tip of a standard pulmonary artery catheter. Thiswavelength-specific optical sensor can monitor the renal functionspecific elimination of a designed optically detectable chemical entity.Thus, by a method substantially analogous to a dye dilution curve,real-time renal function can be monitored by the disappearance of theoptically detected compound. Appropriate modification of a standardpulmonary artery catheter generally includes merely making the fiberoptic sensor wavelength-specific. Catheters that incorporate fiber optictechnology for measuring mixed venous oxygen saturation exist currently.

In an embodiment of this aspect, the present invention provides a methodof monitoring a physiological state or condition of a patient undergoingtreatment. In this method, an effective amount of an optical agent ofthe present invention is administered to a mammal (e.g., a patientundergoing treatment). Further, the optical agent that has beenadministered is exposed to electromagnetic radiation. In addition,electromagnetic radiation transmitted, scattered or emitted by theoptical agent is detected. In some embodiments, a change in thewavelengths or intensities of electromagnetic radiation emitted by theoptical agent that has been administered to the mammal may be detectedand/or measured, optionally as a function of time. Methods of thisaspect of the present invention include in situ, real time methods ofmonitoring renal function in the mammal, wherein the optical agent iscleared by the renal system of the subject.

In an embodiment particularly useful for monitoring physiologicalfunction of an organ or tissue of a subject, the method of this aspectfurther comprises: (i) exposing the detectable agent in the bodily fluidto electromagnetic radiation for exciting emission from the detectableagent; (ii) measuring the emission from the detectable agent that is inthe bodily fluid; and (iii) determining the physiological function ofthe organ or tissue of the subject based on measurement of the emission.The present invention includes fluorescence detection of an agent whichis cleared from the bloodstream by the kidneys. Thus, assessment ofrenal function by in vivo fluorescence detection is encompassed withinthe scope of the invention. The invention can also be used to monitorthe efficiency of hemodialysis. The organ or tissue in some methods is akidney, or tissue or cells thereof, of the subject, wherein the presentinvention provides methods for monitoring renal function of the subject.

Methods of this aspect of the present invention may further comprise avariety of optional steps, including analysis of the measured emissionfrom the optical agent as a function of time, such as over a periodranging from 10 minutes to 48 hours. In an embodiment, for example, themethod further comprises measuring a blood clearance parameter orprofile of the detectable agent administered to the subject. A method ofthis aspect further comprises comparing the blood clearance parameter orprofile of the detectable agent administered to the subject to areference blood clearance parameter or profile. Useful blood clearanceparameters for this aspect of the invention include instantaneous and/oraverage rates of clearance of the detectable agent. A method of thisaspect further comprises comparing the emission from the subject orfunction thereof with one or more emission reference values or afunction thereof of a reference subject. In some embodiments, measuringthe emission from the detectable agent comprises measuring emission fromthe detectable agent in the bodily fluid at a plurality of differenttimes. The clearance of a plurality of separate tracers may bedetermined simultaneously by selecting excitation wavelengths andfilters for the emitted electromagnetic radiation. The concentration vstime or fluorescence intensity vs time curves may be analyzed in realtime by a microprocessor. The resulting clearance rates may becalculated and displayed for immediate clinical impact. In cases whereunlabeled competing compounds are present, a single blood sample may beanalyzed for the concentration of these competing compounds and theresults used to calculate a flux (micromoles/minute) through theclearance pathways.

In accordance with one embodiment of the present invention, a method isdisclosed for determining cell and/or organ function by measuring theblood pool clearance of an optical agent, sometimes referred to hereinas a tracer. The cell and/or organ function can be determined by therate these cells remove the tracer from the bloodstream. Function canalso be assessed by measuring the rate the cells of interest accumulatethe tracer or convert it into an active or other form. The agent may betargeted to a group of cells or organ which is a high capacity clearancesystem. The agent may be an optical agent comprising a pyrazinecompound, or derivative or conjugate thereof including bioconjugate,such as the compositions provided in formulae (FX1)-(FX25). For opticalagents containing a pyrazine component, blood pool clearance may bemeasured using a light source-photodetector device that measures tissueabsorbance or fluorescence in a non-target site, such as an ear lobe,finger, brain or retina. Accumulation of the tracer within the cells ofinterest can be assessed in a similar fashion. The detection of suchaccumulation can be facilitated by using fluorophores which emit in thenear infrared wavelengths since body tissues are relatively transparentat these wavelengths.

The present invention may be used for rapid bedside evaluation ofbiologic functions. For example, data on renal function may be obtainedin less than sixty minutes at the bedside after a single intravenousinjection. In accordance with one embodiment, a patient may receive abolus injection of a plurality (e.g., three) of different compounds,each containing a different optical agent (e.g., fluorophore, dye,chromophore).

In an embodiment, the method comprises exposing the detectable agent inthe bodily fluid to electromagnetic radiation having wavelengthsselected over the range of 350 nm to 1300 nm. Optionally, excitation isachieved using electromagnetic radiation substantially free (e.g., lessthan about 10% of total radiant energy), of ultraviolet radiation forexample to minimize exposure of the subject to electromagnetic radiationcapable of causing unwanted cell or tissue damage. Excitation of opticalagents may be provided by a wide range of techniques and optical sourcesas known in the art, including use of laser, fiber optic and/orendoscopic optical sources and methods. The present invention includesmethods using multiphoton excitation of optical agents. In anembodiment, the method comprises measuring fluorescence from thedetectable agent having wavelengths selected over the range of 350 nm to1300 nm. Detection of emission, including fluorescence, can be achievedby wide a range of techniques and detection systems as known in the art,including detection by eye (e.g., visualization) and two-dimensional orthree-dimensional detection.

Example 18 Pharmaceutical Formulations

Therapeutically Effective Amount

Toxicity and diagnostic efficacy of compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds exhibiting toxic side effects may be used, care should betaken to design a delivery system that minimizes potential damage tocells and reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages for use in humans and othermammals. The dosage of such compounds lies preferably within a range ofcirculating plasma or other bodily fluid concentrations that include theED₅₀ and provides effective imaging results (i.e., ability to image ordiagnose a condition or system). The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound of the present invention, the visuallyeffective amount can be estimated initially from cell culture assays. Adosage may be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful dosages in humans and other mammals.Compound levels in plasma may be measured, for example, by highperformance liquid chromatography.

As used herein, a “bodily fluid” is a liquid originating inside a livingorganism. A bodily fluid can be excretable or secretable from the body.Blood or blood components are particular examples of bodily fluids.

As used herein, “proximate” means near or on. For example, a sensor islocated proximate to a light emission source if the sensor can detectthe light emission. As used herein, “renal function” is a measure of thefunctioning of the renal system or component thereof. The renal systemincludes the kidneys, renal artery, and urinary system.

An amount of a compound that may be combined with a pharmaceuticallyacceptable carrier to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. It willbe appreciated by those skilled in the art that the unit content of acompound contained in an individual dose of each dosage form need not initself constitute effective imaging amount, as the necessary imagingeffective amount could be reached by administration of a number ofindividual doses. The selection of dosage depends upon the dosage formutilized, and the particular purpose to be achieved according to thedetermination of those skilled in the art.

The dosage and dosage regime for imaging a disease or condition may beselected in accordance with a variety of factors, including the type,age, weight, sex, diet and/or medical condition of the patient, theroute of administration, pharmacological considerations such asactivity, efficacy, pharmacokinetic and/or toxicology profiles of theparticular compound employed, whether a compound delivery system isutilized, and/or whether the compound is administered as a pro-drug orpart of a drug combination. Thus, the dosage regime actually employedmay vary widely from subject to subject, or disease to disease anddifferent routes of administration may be employed in different clinicalsettings.

Subjects are preferably animals (e.g., mammals, reptiles and/or avians),more preferably humans, horses, cows, dogs, cats, sheep, pigs, and/orchickens, and most preferably humans.

In an embodiment, the invention provides a medicament which comprises animaging or disgnostically effective amount of one or more compositionsof the invention, such as a compound of any one of formulas(FX1)-(FX25). In an embodiment, the invention provides a method formaking a medicament for imaging or diagnosing of a condition describedherein such as renal failure or renal system disorder. In an embodiment,the invention provides a method for making a medicament for diagnosis oraiding in the diagnosis of a condition described herein. In anembodiment, the invention provides the use of one or more compositionsset forth herein for the making of a medicament.

Compounds of the invention can have prodrug forms. Prodrugs of thecompounds of the invention are useful in embodiments includingcompositions and methods. Any compound that will be converted in vivo toprovide a biologically, pharmaceutically, or diagnostically active formof a compound of the invention is a prodrug. Various examples and formsof prodrugs are well known in the art. Examples of prodrugs are found,inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier,1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K.Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design andDevelopment, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5,“Design and Application of Prodrugs,” by H. Bundgaard, at pp. 113-191,1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38(1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol.77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A BiochemicalApproach, Oxford University Press, New York, pages 388-392). A prodrug,such as a pharmaceutically acceptable prodrug can represent prodrugs ofthe compounds of the invention which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand lower animals without undue toxicity, irritation, allergic response,and the like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use. Prodrugs of the invention can berapidly transformed in vivo to a parent compound of a compound describedherein, for example, by hydrolysis in blood or by other cell, tissue,organ, or system processes. Further discussion is provided in T. Higuchiand V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S.Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press(1987).

The invention contemplates pharmaceutically active compounds eitherchemically synthesized or formed by in vivo biotransformation tocompounds set forth herein.

In an embodiment, a composition of the invention is isolated orpurified. In an embodiment, an isolated or purified compound may be atleast partially isolated or purified as would be understood in the art.In an embodiment, the composition of the invention has a chemical purityof 95%, optionally for some applications 99%, optionally for someapplications 99.9%, optionally for some applications 99.99% pure, andoptionally for some applications 99.999% pure.

Typically, a compound of the invention, or pharmaceutically acceptablesalt thereof, is administered to a subject in a diagnostically orimaging effective amount. One skilled in the art generally can determinean appropriate dosage. Factors affecting a particular dosage regimen(including the amount of compound delivered, frequency ofadministration, and whether administration is continuous orintermittent) include, for example, the type, age, weight, sex, diet,and condition of the subject; the type of pathological condition and itsseverity; and the nature of the desired therapeutic effect.Pharmacological considerations include compound activity, efficacy,pharmacokinetic, and toxicology profiles of the particular compoundused; the route of administration and whether a drug delivery system isutilized; and whether the compound is administered as part of acombination therapy (e.g., whether the agent is administered incombination with one or more other active compounds, other agents,radiation, and the like).

Compositions for oral administration may be, for example, prepared in amanner such that a single dose in one or more oral preparations containsat least about 20 mg of the pyrazine compound per square meter ofsubject body surface area, or at least about 50, 100, 150, 200, 300,400, or 500 mg of the pyrazine compound per square meter of subject bodysurface area (the average body surface area for a human is, for example,1.8 square meters). In particular, a single dose of a composition fororal administration can contain from about 20 to about 600 mg, and incertain aspects from about 20 to about 400 mg, in another aspect fromabout 20 to about 300 mg, and in yet another aspect from about 20 toabout 200 mg of the pyrazine compound per square meter of subject bodysurface area. Compositions for parenteral administration can be preparedin a manner such that a single dose contains at least about 20 mg of thepyrazine compound per square meter of subject body surface area, or atleast about 40, 50, 100, 150, 200, 300, 400, or 500 mg of the pyrazinecompound per square meter of subject body surface area. In particular, asingle dose in one or more parenteral preparations contains from about20 to about 500 mg, and in certain aspects from about 20 to about 400mg, and in another aspect from about 20 to about 450 mg, and in yetanother aspect from about 20 to about 350 mg of the pyrazine compoundper square meter of subject body surface area. It should be recognizedthat these oral and parenteral dosage ranges represent generallypreferred dosage ranges, and are not intended to limit the invention.The dosage regimen actually employed can vary widely, and, therefore,can deviate from the generally preferred dosage regimen. It iscontemplated that one skilled in the art will tailor these ranges to theindividual subject.

It is further contemplated that the pyrazine compounds and salts of thisinvention can be used in the form of a kit that is suitable for use inperforming the methods described herein, packaged in a container. Thekit can contain the pyrazine compounds and, optionally, appropriatediluents, devices or device components suitable for administration andinstructions for use in accordance with the methods of the invention.The devices can include parenteral injection devices, such as syringesor transdermal patch or the like. Device components can includecartridges for use in injection devices and the like. In one aspect, thekit includes a first dosage form including a pyrazine compound or saltof this invention and a second dosage form including another activeingredient in quantities sufficient to carry out the methods of theinvention. The first dosage form and the second dosage form together caninclude a diagnostically effective amount of the compounds for imagingor diagnosing the targeted condition(s).

This invention also is directed, in part, to pharmaceutical compositionsincluding a diagnostically effective amount of a compound or salt ofthis invention, as well as processes for making such compositions. Suchcompositions generally include one or more pharmaceutically acceptablecarriers (e.g., excipients, vehicles, auxiliaries, adjuvants, diluents)and may include other active ingredients. Formulation of thesecompositions may be achieved by various methods known in the art. Ageneral discussion of these methods may be found in, for example,Hoover, John E., Remington's Pharmaceutical Sciences (Mack PublishingCo., Easton, Pa.: 1975). See also, Lachman, L., eds., PharmaceuticalDosage Forms (Marcel Decker, New York, N.Y., 1980).

The preferred composition depends on the route of administration. Anyroute of administration may be used as long as the target of thecompound or pharmaceutically acceptable salt is available via thatroute. Suitable routes of administration include, for example, oral,intravenous, parenteral, inhalation, rectal, nasal, topical (e.g.,transdermal and intraocular), intravesical, intrathecal, enteral,pulmonary, intralymphatic, intracavital, vaginal, transurethral,intradermal, aural, intramammary, buccal, orthotopic, intratracheal,intralesional, percutaneous, endoscopical, transmucosal, sublingual, andintestinal administration.

Pharmaceutically acceptable carriers that may be used in conjunctionwith the compounds of the invention are well known to those of ordinaryskill in the art. Carriers can be selected based on a number of factorsincluding, for example, the particular pyrazine compound(s) orpharmaceutically acceptable salt(s) used; the compound's concentration,stability, and intended bioavailability; the condition being treated;the subject's age, size, and general condition; the route ofadministration; etc. A general discussion related to carriers may befound in, for example, J. G. Nairn, Remington's Pharmaceutical Science,pp. 1492-1517 (A. Gennaro, ed., Mack Publishing Co., Easton, Pa.(1985)).

Solid dosage forms for oral administration include, for example,capsules, tablets, gelcaps, pills, dragees, troches, powders, granules,and lozenges. In such solid dosage forms, the compounds orpharmaceutically acceptable salts thereof can be combined with one ormore pharmaceutically acceptable carriers. The compounds andpharmaceutically acceptable salts thereof can be mixed with carriersincluding, but not limited to, lactose, sucrose, starch powder, cornstarch, potato starch, magnesium carbonate, microcrystalline cellulose,cellulose esters of alkanoic acids, cellulose alkyl esters, talc,stearic acid, magnesium stearate, magnesium oxide, sodium and calciumsalts of phosphoric and sulfuric acids, sodium carbonate, agar,mannitol, sorbitol, sodium saccharin, gelatin, acacia gum, alginic acid,sodium alginate, tragacanth, colloidal silicon dioxide, croscarmellosesodium, polyvinylpyrrolidone, and/or polyvinyl alcohol, and thentableted or encapsulated for convenient administration. Such capsules ortablets can contain a controlled-release formulation, as can be providedin a dispersion of the compound or salt in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formsalso can include buffering agents, such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills additionally can,for example, include a coating (e.g., an enteric coating) to delaydisintegration and absorption. The concentration of the pyrazinecompound in a solid oral dosage form can be from about 5 to about 50%,and in certain aspects from about 8 to about 40%, and in another aspectfrom about 10 to about 30% by weight based on the total weight of thecomposition.

Liquid dosage forms of the compounds of the invention for oraladministration include, for example, pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs containing inertdiluents commonly used in the art (e.g., water). Such compositions alsocan include adjuvants, such as wetting, emulsifying, suspending,flavoring (e.g., sweetening), and/or perfuming agents. The concentrationof the pyrazine compound in the liquid dosage form can be from about0.01 to about 5 mg, and in certain aspects from about 0.01 to about 1mg, and in another aspect from about 0.01 to about 0.5 mg per ml of thecomposition. Low concentrations of the compounds of the invention inliquid dosage form can be prepared in the case that the pyrazinecompound is more soluble at low concentrations. Techniques for makingoral dosage forms useful in the invention are generally described in,for example, Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes,Editors (1979)). See also, Lieberman et al., Pharmaceutical DosageForms: Tablets (1981). See also, Ansel, Introduction to PharmaceuticalDosage Forms (2nd Edition (1976)).

In some aspects of the invention, tablets or powders for oraladministration can be prepared by dissolving the pyrazine compound in apharmaceutically acceptable solvent capable of dissolving the compoundto form a solution and then evaporating when the solution is dried undervacuum. A carrier can also be added to the solution before drying. Theresulting solution can be dried under vacuum to form a glass. The glasscan then mix with a binder to form a powder. This powder may be mixedwith fillers or other conventional tableting agents, and then processedto form a tablet. Alternatively, the powder may be added to a liquidcarrier to form a solution, emulsion, suspension, or the like.

In some aspects, solutions for oral administration are prepared bydissolving the pyrazine compound in a pharmaceutically acceptablesolvent capable of dissolving the compound to form a solution. Anappropriate volume of a carrier is added to the solution while stirringto form a pharmaceutically acceptable solution for oral administration.

“Parenteral administration” includes subcutaneous injections,intravenous injections, intraarterial injections, intraorbitalinjections, intracapsular injections, intraspinal injections,intraperitoneal injections, intramuscular injections, intrasternalinjections, and infusion. Dosage forms suitable for parenteraladministration include solutions, suspensions, dispersions, emulsions,and any other dosage form that can be administered parenterally.

Injectable preparations (e.g., sterile injectable aqueous or oleaginoussuspensions) can be formulated according to the known art using suitabledispersing, wetting agents, and/or suspending agents. Acceptablevehicles for parenteral use include both aqueous and nonaqueouspharmaceutically-acceptable solvents. Suitable pharmaceuticallyacceptable aqueous solvents include, for example, water, salinesolutions, dextrose solutions (e.g., such as DW5), electrolytesolutions, etc.

In one embodiment, the present pyrazine compounds are formulated asnanoparticles or microparticles. Use of such nanoparticle ormicroparticle formulations may be beneficial for some applications toenhance delivery, localization, target specificity, administration, etc.of the pyrazine compound. Potentially useful nanoparticles andmicroparticles include, but are not limited to, micelles, liposomes,microemulsions, nanoemulsions, vesicles, tubular micelles, cylindricalmicelles, bilayers, folded sheets structures, globular aggregates,swollen micelles, inclusion complex, encapsulated droplets,microcapsules, nanocapsules or the like. As will be understood by thosehaving skill in the art, the present pyrazine compounds can be locatedinside the nanoparticle or microparticle, within a membrane or wall ofthe nanoparticle or microparticle, or outside of (but bonded to orotherwise associated with) the nanoparticle or microparticle. The agentformulated in nanoparticles or microparticles may be administered by anyof the routes previously described. In a formulation applied topically,the pyrazine compound is slowly released over time. In an injectableformulation, the liposome, micelle, capsule, etc., circulates in thebloodstream and is delivered to the desired site (e.g., renal system).

Preparation and loading of nanoparticles and microparticles are wellknown in the art. As one example, liposomes may be prepared fromdipalmitoyl phosphatidylcholine (DPPC) or egg phosphatidylcholine (PC)because this lipid has a low heat transition. Liposomes are made usingstandard procedures as known to one skilled in the art (e.g.,Braun-Falco et al., (Eds.), Griesbach Conference, Liposome Dermatics,Springer-Verlag, Berlin (1992), pp. 69 81; 91 117 which is expresslyincorporated by reference herein). Polycaprolactone, poly(glycolic)acid, poly(lactic) acid, polyanhydride or lipids may be formulated asmicrospheres. As an illustrative example, the present pyrazine compoundsmay be mixed with polyvinyl alcohol (PVA), the mixture then dried andcoated with ethylene vinyl acetate, then cooled again with PVA. In aliposome, the present pyrazine compounds may be within one or both lipidbilayers, in the aqueous between the bilayers, or within the center orcore. Liposomes may be modified with other molecules and lipids to forma cationic liposome. Liposomes may also be modified with lipids torender their surface more hydrophilic which increases their circulationtime in the bloodstream. The thus-modified liposome has been termed a“stealth” liposome, or a long-lived liposome, as described in U.S. Pat.No. 6,258,378, and in Stealth Liposomes, Lasic and Martin (Eds.) 1995CRC Press, London, which are expressly incorporated by reference herein.Encapsulation methods include detergent dialysis, freeze drying, filmforming, injection, as known to one skilled in the art and disclosed in,for example, U.S. Pat. No. 6,406,713 which is expressly incorporated byreference herein in its entirety. Optionally, the present compositionsand methods include a micelle delivery system, for example, involvingone or more PEG-based amphiphilic polymers developed for drug deliveryincluding PEG-poly(ε-caprolactone), PEG-poly(amino acid),PEG-polylactide or a PEG-phospholid constructs; a cross linkedpoly(acrylic acid) polymer system, a phospholipid-based system and/orblock copolymer systems comprising one or more of the following polymerblocks a poly(lactic acid) polymer block, a poly(propylene glycol)polymer block; a poly(amino acid) polymer block; a poly(ester) polymerblock; and a poly(ε-caprolactone) polymer block, a poly(ethylene glycol)block, a poly(acrylic acid) block, a polylactide block, a polyesterblock, a polyamide block, a polyanhydride block, a polyurethane block, apolyimine block, a polyurea block, a polyacetal block, a polysaccharideblock and a polysiloxane block.

Suitable pharmaceutically-acceptable nonaqueous solvents include, butare not limited to, the following (as well as mixtures thereof):alcohols (these include, for example, σ-glycerol formal, β-glycerolformal, 1,3-butyleneglycol, aliphatic or aromatic alcohols having from 2to about 30 carbons (e.g., methanol, ethanol, propanol, isopropanol,butanol, t-butanol, hexanol, octanol, amylene hydrate, benzyl alcohol,glycerin (glycerol), glycol, hexylene, glycol, tetrahydrofuranylalcohol, cetyl alcohol, and stearyl alcohol), fatty acid esters of fattyalcohols (e.g., polyalkylene glycols, such as polypropylene glycol andpolyethylene glycol), sorbitan, sucrose, and cholesterol); amides (theseinclude, for example, dimethylacetamide (DMA), benzyl benzoate DMA,dimethylformamide, N-hydroxyethyl-O-lactamide,N,N-dimethylacetamide-amides, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone,and polyvinylpyrrolidone); esters (these include, for example, acetateesters (e.g., monoacetin, diacetin, and triacetin), aliphatic andaromatic esters (e.g., ethyl caprylate or octanoate, alkyl oleate,benzyl benzoate, or benzyl acetate), dimethylsulfoxide (DMSO), esters ofglycerin (e.g., mono, di, and tri-glyceryl citrates and tartrates),ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyloleate, fatty acid esters of sorbitan, glyceryl monostearate, glycerideesters (e.g., mono, di, or tri-glycerides), fatty acid esters (e.g.,isopropyl myristrate), fatty acid derived PEG esters (e.g.,PEG-hydroxyoleate and PEG-hydroxystearate), N-methyl pyrrolidinone,pluronic 60, polyoxyethylene sorbitol oleic polyesters (e.g.,poly(ethoxylated)₃₀₋₆₀ sorbitol poly(oleate)₂₋₄, poly(oxyethylene)₁₅₋₂₀monooleate, poly(oxyethylene)₁₅₋₂₀ mono 12-hydroxystearate, andpoly(oxyethylene)₁₅₋₂₀ mono ricinoleate), polyoxyethylene sorbitanesters (e.g., polyoxyethylene-sorbitan monooleate,polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitanmonolaurate, polyoxyethylene-sorbitan monostearate, and POLYSORBATE 20,40, 60, and 80 (from ICI Americas, Wilmington, Del.)),polyvinylpyrrolidone, alkyleneoxy modified fatty acid esters (e.g.,polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oils,such as CREMOPHOR EL solution or CREMOPHOR RH 40 solution), saccharidefatty acid esters (i.e., the condensation product of a monosaccharide(e.g., pentoses, such as, ribose, ribulose, arabinose, xylose, lyxose,and xylulose; hexoses, such as glucose, fructose, galactose, mannose,and sorbose; trioses; tetroses; heptoses; and octoses), disaccharide(e.g., sucrose, maltose, lactose, and trehalose), oligosaccharide, or amixture thereof with one or more C₄-C₂₂ fatty acids (e.g., saturatedfatty acids, such as caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, and stearic acid; and unsaturated fatty acids, suchas palmitoleic acid, oleic acid, elaidic acid, erucic acid, and linoleicacid), and steroidal esters); ethers (these are typically alkyl, aryl,and cyclic ethers having from 2 to about 30 carbons. Examples includediethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycolmonoethyl ether), and glycofurol (tetrahydrofurfuranyl alcoholpolyethylene glycol ether); ketones (these typically have from about 3to about 30 carbons. Examples include acetone, methyl ethyl ketone,methyl isobutyl ketone); hydrocarbons (these are typically aliphatic,cycloaliphatic, and aromatic hydrocarbons having from about 4 to about30 carbons). Examples include benzene, cyclohexane, dichloromethane,dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolane,tetramethylenesulfone, tetramethylenesulfoxide, toluene,dimethylsulfoxide (DMSO); and tetramethylene sulfoxide; oils (theseinclude oils of mineral, vegetable, animal, essential, or syntheticorigin). These include mineral oils, such as aliphatic and wax-basedhydrocarbons, aromatic hydrocarbons, mixed aliphatic and aromatic basedhydrocarbons, and refined paraffin oil; vegetable oils, such as linseed,tung, safflower, soybean, castor, cottonseed, groundnut, rapeseed,coconut, palm, olive, corn, corn germ, sesame, persic, and peanut oil;glycerides, such as mono-, di-, and triglycerides; animal oils, such asfish, marine, sperm, cod-liver, haliver, squaiene, squalane, and sharkliver oil; oleic oils; and polyoxyethylated castor oil); alkyl, alkenyl,or aryl halides (these include alkyl or aryl halides having from 1 toabout 30 carbons and one or more halogen substituents. Examples includemethylene chloride); monoethanolamine; petroleum benzin; trolamine;omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid,eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid);polyglycol ester of 12-hydroxystearic acid and polyethylene glycol(SOLUTOL HS-15, from BASF, Ludwigshafen, Germany); polyoxyethyleneglycerol; sodium laurate; sodium oleate; and sorbitan monooleate. Otherpharmaceutically acceptable solvents for use in the invention are wellknown to those of ordinary skill in the art. General discussion relatingto such solvents may be found in, for example, The Chemotherapy SourceBook (Williams & Wilkens Publishing), The Handbook of PharmaceuticalExcipients, (American Pharmaceutical Association, Washington, D.C., andThe Pharmaceutical Society of Great Britain, London, England, 1968),Modern Pharmaceutics 3d ed., (G. Banker et. al., eds., Marcel Dekker,Inc., New York, N.Y. (1995)), The Pharmacological Basis of Therapeutics,(Goodman & Gilman, McGraw Hill Publishing), Pharmaceutical Dosage Forms,(H. Lieberman et. al., eds., Marcel Dekker, Inc., New York, N.Y.(1980)), Remington's Pharmaceutical Sciences, 19th ed., (A. Gennaro,ed., Mack Publishing, Easton, Pa., (1995)), The United StatesPharmacopeia 24, The National Formulary 19, (National Publishing,Philadelphia, Pa. (2000)); Spiegel, A. J., et al., “Use of NonaqueousSolvents in Parenteral Products,” J. Pharma. Sciences, Vol. 52, No. 10,pp. 917-927 (1963).

Solvents useful in the invention include, but are not limited to, thoseknown to stabilize the pyrazine compounds or pharmaceutically acceptablesalts thereof. These typically include, for example, oils rich intriglycerides, such as safflower oil, soybean oil, and mixtures thereof;and alkyleneoxy-modified fatty acid esters, such as polyoxyl 40hydrogenated castor oil and polyoxyethylated castor oils (e.g.,CREMOPHOR EL solution or CREMOPHOR RH 40 solution). Commerciallyavailable triglycerides include INTRALIPID emulsified soybean oil(Kabi-Pharmacia Inc., Stockholm, Sweden), NUTRALIPID emulsion (McGaw,Irvine, Calif.), LIPOSYN II 20% emulsion (a 20% fat emulsion solutioncontaining 100 mg safflower oil, 100 mg soybean oil, 12 mg eggphosphatides, and 25 mg glycerin per ml of solution; AbbottLaboratories, Chicago, Ill.), LIPOSYN III 2% emulsion (a 2% fat emulsionsolution containing 100 mg safflower oil, 100 mg soybean oil, 12 mg eggphosphatides, and 25 mg glycerin per ml of solution; AbbottLaboratories, Chicago, Ill.), natural or synthetic glycerol derivativescontaining the docosahexaenoyl group at levels of from about 25 to about100% (by weight based on the total fatty acid content) (DHASCO fromMartek Biosciences Corp., Columbia, Md.; DHA MAGURO from DaitoEnterprises, Los Angeles, Calif.; SOYACAL; and TRAVEMULSION). Ethanol inparticular is a useful solvent for dissolving a pyrazine compound orpharmaceutically acceptable salt thereof to form solutions, emulsions,and the like.

Additional components can be included in the compositions of thisinvention for various purposes generally known in the pharmaceuticalindustry. These components tend to impart properties that, for example,enhance retention of the pyrazine compound or salt at the site ofadministration, protect the stability of the composition, control thepH, and facilitate processing of the pyrazine compound or salt intopharmaceutical formulations, and the like. Specific examples of suchcomponents include cryoprotective agents; agents for preventingreprecipitation of the pyrazine compound or salt surface; active,wetting, or emulsifying agents (e.g., lecithin, polysorbate-80, TWEEN80, pluronic 60, and polyoxyethylene stearate); preservatives (e.g.,ethyl-p-hydroxybenzoate); microbial preservatives (e.g., benzyl alcohol,phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal, and paraben);agents for adjusting pH or buffering agents (e.g., acids, bases, sodiumacetate, sorbitan monolaurate, etc.); agents for adjusting osmolarity(e.g., glycerin); thickeners (e.g., aluminum monostearate, stearic acid,cetyl alcohol, stearyl alcohol, guar gum, methyl cellulose,hydroxypropylcellulose, tristearin, cetyl wax esters, polyethyleneglycol, etc.); colorants; dyes; flow aids; non-volatile silicones (e.g.,cyclomethicone); clays (e.g., bentonites); adhesives; bulking agents;flavorings; sweeteners; adsorbents; fillers (e.g., sugars such aslactose, sucrose, mannitol, sorbitol, cellulose, calcium phosphate,etc.); diluents (e.g., water, saline, electrolyte solutions, etc.);binders (e.g., gelatin; gum tragacanth; methyl cellulose; hydroxypropylmethylcellulose; sodium carboxymethyl cellulose; polyvinylpyrrolidone;sugars; polymers; acacia; starches, such as maize starch, wheat starch,rice starch, and potato starch; etc.); disintegrating agents (e.g.,starches, such as maize starch, wheat starch, rice starch, potatostarch, and carboxymethyl starch; cross-linked polyvinyl pyrrolidone;agar; alginic acid or a salt thereof, such as sodium alginate;croscarmellose sodium; crospovidone; etc); lubricants (e.g., silica;talc; stearic acid and salts thereof, such as magnesium stearate;polyethylene glycol; etc.); coating agents (e.g., concentrated sugarsolutions including gum arabic, talc, polyvinyl pyrrolidone, carbopolgel, polyethylene glycol, titanium dioxide, etc.); and antioxidants(e.g., sodium metabisulfite, sodium bisulfite, sodium sulfite, dextrose,phenols, thiophenols, etc.). Techniques and compositions for makingparenteral dosage forms are generally known in the art. Formulations forparenteral administration may be prepared from one or more sterilepowders and/or granules having a compound or salt of this invention andone or more of the carriers or diluents mentioned for use in theformulations for oral administration. The powder or granule typically isadded to an appropriate volume of a solvent (typically while agitating(e.g., stirring) the solvent) that is capable of dissolving the powderor granule. Particular solvents useful in the invention include, forexample, water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers.

Emulsions for parenteral administration can be prepared by, for example,dissolving a compound or salt of this invention in any pharmaceuticallyacceptable solvent capable of dissolving the compound to form asolution; and adding an appropriate volume of a carrier to the solutionwhile stirring to form the emulsion. Solutions for parenteraladministration can be prepared by, for example, dissolving a compound orsalt of this invention in any pharmaceutically acceptable solventcapable of dissolving the compound to form a solution; and adding anappropriate volume of a carrier to the solution while stirring to formthe solution.

Suppositories for rectal administration can be prepared by, for example,mixing the drug with a suitable nonirritating excipient that is solid atordinary temperatures, but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Suitable excipientsinclude, for example, cocoa butter; synthetic mono-, di-, ortriglycerides; fatty acids; and/or polyethylene glycols.

“Topical administration” includes the use of transdermal administration,such as transdermal patches or iontophoresis devices.

If desired, the emulsions or solutions described above for oral orparenteral administration can be packaged in IV bags, vials, or otherconventional containers in concentrated form, and then diluted with apharmaceutically acceptable liquid (e.g., saline) to form an acceptablepyrazine compound concentration before use.

It is understood that this invention is not limited to the particularcompounds, methodology, protocols, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the invention which will be limited onlyby the appended claims.

Compositions of the invention include formulations and preparationscomprising one or more of the present compounds provided in an aqueoussolution, such as a pharmaceutically acceptable formulation orpreparation. Optionally, compositions of the invention further compriseone or more pharmaceutically acceptable surfactants, buffers,electrolytes, salts, carriers, binders, coatings, preservatives and/orexcipients.

Formulations and Use

Compounds and bioconjugates of the present invention may be formulatedby known methods for administration to a subject using several routeswhich include, but are not limited to, parenteral, oral, topical,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and ophthalmic routes. An individualcompound/bioconjugate may be administered in combination with one ormore additional compounds/bioconjugates of the present invention and/ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the compound(s)/bioconjugate(s) or attached to thecompound(s)/bioconjugate(s) by ionic, covalent, Van der Waals,hydrophobic, hydrophilic or other physical forces. Administration mayoptionally be localized in a subject. Administration may optionally besystemic.

Compounds and bioconjugates of the present invention may be formulatedby any conventional manner using one or more pharmaceutically acceptablecarriers. Thus, the compounds/bioconjugates and their pharmaceuticallyacceptable salts and solvates may be specifically formulated foradministration, e.g., by inhalation or insufflation (either through themouth or the nose) or oral, buccal, parenteral or rectal administration.The compounds/bioconjugates may take the form of charged, neutral and/orother pharmaceutically acceptable salt forms. Examples ofpharmaceutically acceptable carriers include, but are not limited to,those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A. R. Gennaro,Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

Compounds and bioconjugates of the present invention may be formulatedin the form of solutions, suspensions, emulsions, tablets, pills,capsules, powders, controlled- or sustained-release formulations and thelike. Such formulations will contain a therapeutically effective amountof the compound/bioconjugate, preferably in purified form, together witha suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

Parenteral Administration

Compounds and bioconjugates of the present invention may be formulatedfor parenteral administration by injection (e.g., by bolus injection orcontinuous infusion). Formulations for injection may be presented inunit dosage form in ampoules or in multi-dose containers with anoptional preservative added. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass,plastic or the like. The formulation may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

For example, a parenteral preparation may be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent (e.g., as a solution in 1,3-butanediol). Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid may be used inthe parenteral preparation.

Alternatively, compounds and bioconjugates of the present invention maybe formulated in powder form for constitution with a suitable vehicle,such as sterile pyrogen-free water, before use. For example, acompound/bioconjugate suitable for parenteral administration may includea sterile isotonic saline solution containing between 0.1 percent and 90percent weight per volume of the compound/bioconjugate. By way ofexample, a solution may contain from about 5 percent to about 20percent, more preferably from about 5 percent to about 17 percent, morepreferably from about 8 to about 14 percent, and still more preferablyabout 10 percent of the compound/bioconjugate. The solution or powderpreparation may also include a solubilizing agent and a local anestheticsuch as lignocaine to ease pain at the site of the injection. Othermethods of parenteral delivery of compounds/bioconjugates will be knownto the skilled artisan and are within the scope of the invention.

Oral Administration

For oral administration, a compound/bioconjugate of the invention may beformulated to take the form of tablets or capsules prepared byconventional means with one or more pharmaceutically acceptable carriers(e.g., excipients such as binding agents, fillers, lubricants anddisintegrants):

A. Binding Agents

Binding agents include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.Suitable forms of microcrystalline cellulose include, for example, thematerials sold as AVICEL-PH-101, AVICEL-PH-103 and AVICEL-PH-105(available from FMC Corporation, American Viscose Division, AvicelSales, Marcus Hook, Pa., USA). An exemplary suitable binder is a mixtureof microcrystalline cellulose and sodium carboxymethyl cellulose sold asAVICEL RC-581 by FMC Corporation.

B. Fillers

Fillers include, but are not limited to, talc, calcium carbonate (e.g.,granules or powder), lactose, microcrystalline cellulose, powderedcellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch,pre-gelatinized starch, and mixtures thereof.

C. Lubricants

Lubricants include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, electromagnetic radiation mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md., USA), a coagulated aerosol of synthetic silica (marketed by DeaussaCo. of Plano, Tex., USA), CAB-O-SIL (a pyrogenic silicon dioxide productsold by Cabot Co. of Boston, Mass., USA), and mixtures thereof.

D. Disintegrants

Disintegrants include, but are not limited to, agar-agar, alginic acid,calcium carbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums, and mixtures thereof.

The tablets or capsules may optionally be coated by methods well knownin the art. If binders and/or fillers are used with acompound/bioconjugate of the invention, they are typically formulated asabout 50 to about 99 weight percent of the compound/bioconjugate. In oneaspect, about 0.5 to about 15 weight percent of disintegrant, andparticularly about 1 to about 5 weight percent of disintegrant, may beused in combination with the compound. A lubricant may optionally beadded, typically in an amount of less than about 1 weight percent of thecompound/bioconjugate. Techniques and pharmaceutically acceptableadditives for making solid oral dosage forms are described in Marshall,SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker and Rhodes, Eds.),7:359-427 (1979). Other less typical formulations are known in the art.

Liquid preparations for oral administration may take the form ofsolutions, syrups or suspensions. Alternatively, the liquid preparationsmay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and/or preservatives (e.g.,methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparationsmay also contain buffer salts, flavoring, coloring, perfuming andsweetening agents as appropriate. Preparations for oral administrationmay also be formulated to achieve controlled release of thecompound/bioconjugate. Oral formulations preferably contain 10% to 95%compound/bioconjugate. In addition, a compound/bioconjugate of thepresent invention may be formulated for buccal administration in theform of tablets or lozenges formulated in a conventional manner. Othermethods of oral delivery of compounds/bioconjugates of the inventionwill be known to the skilled artisan and are within the scope of theinvention.

Controlled-Release Administration

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of a compound/bioconjugate and reduce dosagefrequency. Controlled-release preparations can also be used to effectthe time of onset of action or other characteristics, such as bloodlevels of the compound/bioconjugate, and consequently affect theoccurrence of side effects.

Controlled-release preparations may be designed to initially release anamount of a compound/bioconjugate that produces the desired therapeuticeffect, and gradually and continually release other amounts of thecompound/bioconjugate to maintain the level of therapeutic effect overan extended period of time. In order to maintain a near-constant levelof a compound/bioconjugate in the body, the compound/bioconjugate can bereleased from the dosage form at a rate that will replace the amount ofcompound/bioconjugate being metabolized and/or excreted from the body.The controlled-release of a compound/bioconjugate may be stimulated byvarious inducers, e.g., change in pH, change in temperature, enzymes,water, and/or other physiological conditions or molecules.

Controlled-release systems may include, for example, an infusion pumpwhich may be used to administer the compound/bioconjugate in a mannersimilar to that used for delivering insulin or chemotherapy to the bodygenerally, or to specific organs or tumors. Typically, using such asystem, the compound/bioconjugate is administered in combination with abiodegradable, biocompatible polymeric implant that releases thecompound/bioconjugate over a controlled period of time at a selectedsite. Examples of polymeric materials include polyanhydrides,polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinylacetate, and copolymers and combinations thereof. In addition, acontrolled release system can be placed in proximity of a therapeutictarget (e.g., organ, tissue, or group of cells), thus requiring only afraction of a systemic dosage.

Compounds/bioconjugates of the invention may be administered by othercontrolled-release means or delivery devices that are well known tothose of ordinary skill in the art. These include, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or the like, or a combination of any of theabove to provide the desired release profile in varying proportions.Other methods of controlled-release delivery of compounds/bioconjugateswill be known to the skilled artisan and are within the scope of theinvention.

Inhalation Administration

Compounds/bioconjugates of the invention may be administered directly tothe lung of a patient/subject by inhalation. For administration byinhalation, a compound/bioconjugate may be conveniently delivered to thelung by a number of different devices. For example, a Metered DoseInhaler (“MDI”) which utilizes canisters that contain a suitable lowboiling point propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas may be used to deliver a compound/bioconjugatedirectly to the lung. MDI devices are available from a number ofsuppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, ForestLaboratories, Glaxo-Wellcome, Schering Plough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device may be used toadminister a compound/bioconjugate to the lung. DPI devices typicallyuse a mechanism such as a burst of gas to create a cloud of dry powderinside a container, which may then be inhaled by the patient. DPIdevices are also well known in the art and may be purchased from anumber of vendors which include, for example, Fisons, Glaxo-Wellcome,Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. Apopular variation is the multiple dose DPI (“MDDPI”) system, whichallows for the delivery of more than one therapeutic dose. MDDPI devicesare available from companies such as AstraZeneca, GlaxoWellcome, IVAX,Schering Plough, SkyePharma and Vectura. For example, capsules andcartridges of gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound/bioconjugate and asuitable powder base such as lactose or starch for these systems.

Another type of device that may be used to deliver acompound/bioconjugate to the lung is a liquid spray device supplied, forexample, by Aradigm Corporation. Liquid spray systems use extremelysmall nozzle holes to aerosolize liquid compound/bioconjugateformulations that may then be directly inhaled into the lung. Forexample, a nebulizer device may be used to deliver acompound/bioconjugate to the lung. Nebulizers create aerosols fromliquid compound/bioconjugate formulations by using, for example,ultrasonic energy to form fine particles that may be readily inhaled.Examples of nebulizers include devices supplied by Sheffield/SystemicPulmonary Delivery Ltd., Aventis and Batelle Pulmonary Therapeutics.

In another example, an electrohydrodynamic (“EHD”) aerosol device may beused to deliver a compound/bioconjugate to the lung. EHD aerosol devicesuse electrical energy to aerosolize liquid compound/bioconjugatesolutions or suspensions. The electrochemical properties of thecompound/bioconjugate formulation are important parameters to optimizewhen delivering this compound/bioconjugate to the lung with an EHDaerosol device. Such optimization is routinely performed by one of skillin the art. Other methods of intra-pulmonary delivery ofcompounds/bioconjugates will be known to the skilled artisan and arewithin the scope of the invention.

Liquid compound/bioconjugate formulations suitable for use withnebulizers and liquid spray devices and EHD aerosol devices willtypically include the compound/bioconjugate with a pharmaceuticallyacceptable carrier. In one exemplary embodiment, the pharmaceuticallyacceptable carrier is a liquid such as alcohol, water, polyethyleneglycol or a perfluorocarbon. Optionally, another material may be addedto alter the aerosol properties of the solution or suspension of thecompound/bioconjugate. For example, this material may be a liquid suchas an alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid compound/bioconjugate solutions or suspensionssuitable for use in aerosol devices are known to those of skill in theart.

Depot Administration

A compound/bioconjugate of the invention may be formulated as a depotpreparation. Such long-acting formulations may be administered byimplantation (e.g., subcutaneously or intramuscularly) or byintramuscular injection. Accordingly, the compound/bioconjugate may beformulated with suitable polymeric or hydrophobic materials such as anemulsion in an acceptable oil or ion exchange resins, or as sparinglysoluble derivatives such as a sparingly soluble salt. Other methods ofdepot delivery of compounds/bioconjugates will be known to the skilledartisan and are within the scope of the invention.

Topical Administration

For topical application, a compound/bioconjugate may be combined with apharmaceutically acceptable carrier so that an effective dosage isdelivered, based on the desired activity ranging from an effectivedosage, for example, of 1.0 μM to 1.0 mM. In one aspect of theinvention, a topical formulation of a compound/bioconjugate can beapplied to the skin. The pharmaceutically acceptable carrier may be inthe form of, for example, and not by way of limitation, an ointment,cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.

A topical formulation may include a therapeutically effective amount ofa compound/bioconjugate in an ophthalmologically acceptable excipientsuch as buffered saline, mineral oil, vegetable oils such as corn orarachis oil, petroleum jelly, Miglyol 182, alcohol solutions, orliposomes or liposome-like products. Any of these formulations of suchcompounds/bioconjugates may include preservatives, antioxidants,antibiotics, immunosuppressants, and other biologically orpharmaceutically effective agents that do not exert a significantdetrimental effect on the compound/bioconjugate. Other methods oftopical delivery of compounds/bioconjugates will be known to the skilledartisan and are within the scope of the invention.

Rectal Administration

Compounds/bioconjugates of the invention may be formulated in rectalformulations such as suppositories or retention enemas that includeconventional suppository bases such as cocoa butter or other glyceridesand/or binders and/or carriers such as triglycerides, microcrystallinecellulose, gum tragacanth or gelatin. Rectal formulations can contain acompound/bioconjugate in the range of 0.5% to 10% by weight. Othermethods of rectal delivery of compounds/bioconjugates will be known tothe skilled artisan and are within the scope of the invention.

Other Systems of Administration

Various other delivery systems are known in the art and can be used toadminister the compounds/bioconjugates of the invention. Moreover, theseand other delivery systems may be combined and/or modified to promoteoptimization of the administration of compounds/bioconjugates of thepresent invention. Exemplary formulations that includecompounds/bioconjugates of the present invention are described below(the compounds/bioconjugates of the present invention are indicated asthe active ingredient, but those of skill in the art will recognize thatpro-drugs and compound combinations are also meant to be encompassed bythis term):

Kits

Various embodiments of the present invention include kits. Such kits caninclude a compound/bioconjugate of the present invention, optionally oneor more ingredients for preparing a pharmaceutically acceptableformulation of the compound/bioconjugate, and instructions for use(e.g., administration). When supplied as a kit, different components ofa compound/bioconjugate formulation can be packaged in separatecontainers and admixed immediately before use. Such packaging of thecomponents separately can, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the compound/bioconjugate. The pack may, for example,comprise metal or plastic foil such as a blister pack. Such packaging ofthe components separately can also, in certain instances, permitlong-term storage without losing activity of the components. Inaddition, if more than one route of administration is intended or morethan one schedule for administration is intended, the differentcomponents can be packaged separately and not mixed prior to use. Invarious embodiments, the different components can be packaged in onecombination for administration together.

Kits may include reagents in separate containers such as, for example,sterile water or saline to be added to a lyophilized active componentpackaged separately. For example, sealed glass ampules may containlyophilized compounds and in a separate ampule, sterile water, sterilesaline or sterile each of which has been packaged under a neutralnon-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, ceramic, metal or any other material typically employed tohold reagents. Other examples of suitable containers include bottlesthat may be fabricated from similar substances as ampules, and envelopesthat may consist of foil-lined interiors, such as aluminum or an alloy.Other containers include test tubes, vials, flasks, bottles, syringes,and the like. Containers may have a sterile access port, such as abottle having a stopper that can be pierced by a hypodermic injectionneedle. Other containers may have two compartments that are separated bya readily removable membrane that upon removal permits the components tomix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit, orsupplied as electronic mail.

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Choyke, H. A. Austin, and J. A. Frank. Hydrated clearance    of gadolinium-DTPA as a measurement of glomerular filtration rate.    Kidney International 1992, 41, 1595-1598.-   17. N. Lewis, R. Kerr, and C. Van Buren. Comparative evaluation of    urographic contrast media, inulin, and 99mTc-DTPA clearance methods    for determination of glomerular filtration rate in clinical    transplantation. Transplantation 1989, 48, 790-796.-   18. W. N. Tauxe. Tubular Function. In Nuclear Medicine in Clinical    Urology and Nephrology, W. N. Tauxe and E. V. Dubovsky, Editors, pp.    77-105, Appleton Century Crofts: East Norwalk, 1985.-   19. A. R. Fritzberg at al. Mercaptoacetylglycylglycyglycine. Journal    of Nuclear Medicine 1986, 27, 111-120.-   20. G. Ekanoyan and N. W. Levin. In Clinical Practice Guidelines for    Chronic Kidney Disease: Evaluation, Classification, and    Stratification (K/DOQI). National Kidney Foundation: Washington,    D.C. 2002, pp. 1-22.-   21. Ozaki, H. et al. 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STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references cited throughout this application, for example patentdocuments including issued or granted patents or equivalents; patentapplication publications; and non-patent literature documents or othersource material; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although theinvention has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.The specific embodiments provided herein are examples of usefulembodiments of the invention and it will be apparent to one skilled inthe art that the invention may be carried out using a large number ofvariations of the devices, device components, methods steps set forth inthe present description. As will be obvious to one of skill in the art,methods and devices useful for the present methods can include a largenumber of optional composition and processing elements and steps.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. When a compound is described herein suchthat a particular isomer, enantiomer or diastereomer of the compound isnot specified, for example, in a formula or in a chemical name, thatdescription is intended to include each isomers and enantiomer of thecompound described individual or in any combination. Additionally,unless otherwise specified, all isotopic variants of compounds disclosedherein are intended to be encompassed by the disclosure. For example, itwill be understood that any one or more hydrogens in a moleculedisclosed can be replaced with deuterium or tritium. Isotopic variantsof a molecule are generally useful as standards in assays for themolecule and in chemical and biological research related to the moleculeor its use. Methods for making such isotopic variants are known in theart. Specific names of compounds are intended to be exemplary, as it isknown that one of ordinary skill in the art can name the same compoundsdifferently. It is also understood that there are different ways tochemically depict compounds, for example, grouping certain substituentssuch as PEG groups.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds herein, one of ordinary skill in the art can select fromamong a wide variety of available counterions those that are appropriatefor preparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

In some embodiments, a liposome or micelle may be utilized as a carrieror vehicle for the composition.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

The present compositions, preparations and formulations can beformulated into diagnostic compositions for enteral, parenteral,topical, aerosol, inhalation, or cutaneous administration. Topical orcutaneous delivery of the compositions, preparations and formulationsmay also include aerosol formulation, creams, gels, solutions, etc. Thepresent compositions, preparations and formulations are administered indoses effective to achieve the desired diagnostic and/or therapeuticeffect. Such doses may vary widely depending upon the particularcompositions employed in the composition, the organs or tissues to beexamined, the equipment employed in the clinical procedure, the efficacyof the treatment achieved, and the like. These compositions,preparations and formulations contain an effective amount of thecomposition(s), along with conventional pharmaceutical carriers andexcipients appropriate for the type of administration contemplated.These compositions, preparations and formulations may also optionallyinclude stabilizing agents and skin penetration enhancing agents.

Methods of this invention comprise the step of administering an“effective amount” of the present diagnostic compositions, formulationsand preparations containing the present compounds, to diagnosis, image,monitor, or evaluate a biological condition and/or disease state in apatient. The term “effective amount,” as used herein, refers to theamount of the diagnostic formulation, that, when administered to theindividual is effective diagnosis, image, monitor, or evaluate abiological condition and/or disease state. As is understood in the art,the effective amount of a given composition or formulation will dependat least in part upon, the mode of administration (e.g. intravenous,oral, topical administration), any carrier or vehicle employed, and thespecific individual to whom the formulation is to be administered (age,weight, condition, sex, etc.). The dosage requirements needed to achievethe “effective amount” vary with the particular formulations employed,the route of administration, and clinical objectives. Based on theresults obtained in standard pharmacological test procedures, projecteddaily dosages of active compound can be determined as is understood inthe art.

Any suitable form of administration can be employed in connection withthe diagnostic formulations of the invention. The diagnosticformulations of this invention can be administered intravenously, inoral dosage forms, intraperitoneally, subcutaneously, orintramuscularly, all using dosage forms well known to those of ordinaryskill in the pharmaceutical arts.

The diagnostic formulations of this invention can be administered alone,but may be administered with a pharmaceutical carrier selected upon thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The diagnostic formulations of this invention and medicaments of thisinvention may further comprise one or more pharmaceutically acceptablecarrier, excipient, buffer, emulsifier, surfactant, electrolyte ordiluent. Such compositions and medicaments are prepared in accordancewith acceptable pharmaceutical procedures, such as, for example, thosedescribed in Remingtons Pharmaceutical Sciences, 17th edition, ed.Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985).

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. As used herein, ranges specifically include the valuesprovided as endpoint values of the range. For example, a range of 1 to100 specifically includes the end point values of 1 and 100. It will beunderstood that any subranges or individual values in a range orsubrange that are included in the description herein can be excludedfrom the claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

What is claimed is:
 1. A method for use in an optical imaging, medicalimaging, diagnostic, visualization, monitoring, surgical, biomedical ortherapeutic procedure, wherein the method comprises: (i) administeringan effective amount of a renally excretable compound of the formula(FX1):

wherein: R¹ and R³ are each independently —H, —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵,—(CH₂CH₂O)_(b)R⁵, or —(CH₂)_(a)Y¹; each Y¹ is independently —OR⁶,—(CHOH)_(c)R⁷, —NR⁸R⁹, —NHCO(CHOH)_(c)R⁷ or—NHCO(CH₂)_(a)(CH₂CH₂O)_(b)R⁵; each of R², R⁴, R⁵, R⁶ and R⁷ areindependently —H or C₁-C₆ alkyl; R⁸ and R⁹ are independently —H, C₁-C₃alkyl, —(CH₂)_(a)(CHOH)_(c)R⁷, or —(CH₂)_(a)(CH₂CH₂O)_(b)R⁵; each a andc is independently an integer selected from the range of 0 to 6; each bis independently an integer selected from the range of 1 to 120; each pand q is independently an integer selected from the range of 0 to 120;each of m and n is independently an integer selected from the range of 3to 6; (ii) exposing a tissue of the subject's renal system having theadministered compound to electromagnetic radiation, thereby generatingemitted electromagnetic radiation from the compound; and (iii) detectingthe emitted electromagnetic radiation from the compound, therebyvisualizing or imaging at least a portion of the renal system of thesubject.
 2. The method of claim 1, wherein the procedure comprisesdetermining if the administered compound is substantially retained intissue of the subject's renal system.
 3. The method of claim 1, whereinthe compound has plasma binding of less than 10%.
 4. The method of claim1, wherein the biomedical procedure assesses physiological function ofan organ, tissue or system.
 5. The method of claim 1, wherein the atleast a portion of the renal system comprises a ureter, bladder orurethra of the subject.
 6. The method of claim 1, wherein the compoundis of the formula (FX2), (FX3), (FX4), (FX5), or (FX6):


7. The method of claim 1, wherein: each R⁵ is independently C₁-C₃ alkyl;each b is independently an integer from 2 to 50; and m and n are eachindependently 3 or
 4. 8. The method of claim 1, wherein the compound isof the formula (FX7), (FX8), (FX9), (FX10), (FX11), (FX12), (FX13),(FX14), (FX15), or (FX16) or (FX17):


9. The method of claim 1, wherein the compound is of the formula (FX18):

wherein d and h are independently integers selected from the range of 1to
 120. 10. The method of claim 1, wherein each d and h areindependently integers selected from the range of 2 to 50, wherein eacha is 2, and wherein R⁸ and R⁹ are each —(CH₂)_(a)(CHOH)_(c)R⁷.
 11. Themethod of claim 1, wherein the compound is of the formula (FX19):

wherein d and h are independently integers selected from the range of 1to
 120. 12. The method of claim 1, wherein each d and h areindependently integers selected from the range of 2 to 50 or the rangeof 2 to
 24. 13. The method of claim 1, wherein Y¹ is —NR⁸R⁹ and whereinR⁸ and R⁹ are each —(CH₂)_(a)(CHOH)_(c)R⁷ and wherein a is 1 or 2 and cis 2, 3, 4, 5 or
 6. 14. The method of claim 1, wherein the compound isof the formula (FX20) or (FX21):

wherein d and h are independently integers selected from the range of 1to 120.